Primary Stem Research Articles

Investigation of Persistent Photoconductivity of Gallium Nitride Semiconductor and Differentiation of Primary Neural Stem Cells.

A gallium nitride (GaN) semiconductor is one of the most promising materials integrated into biomedical devices to play the roles of connecting, monitoring, and manipulating the activity of biological components, due to its excellent photoelectric properties, chemical stability, and biocompatibility. In this work, it was found that the photogenerated free charge carriers of the GaN substrate, as an exogenous stimulus, served to promote neural stem cells (NSCs) to differentiate into neurons. This was observed through the systematic investigation of the effect of the persistent photoconductivity (PPC) of GaN on the differentiation of primary NSCs from the embryonic rat cerebral cortex. NSCs were directly cultured on the GaN surface with and without ultraviolet (UV) irradiation, with a control sample consisting of tissue culture polystyrene (TCPS) in the presence of fetal bovine serum (FBS) medium. Through optical microscopy, the morphology showed a greater number of neurons with the branching structures of axons and dendrites on GaN with UV irradiation. The immunocytochemical results demonstrated that GaN with UV irradiation could promote the NSCs to differentiate into neurons. Western blot analysis showed that GaN with UV irradiation significantly upregulated the expression of two neuron-related markers, βIII-tubulin (Tuj-1) and microtubule-associated protein 2 (MAP-2), suggesting that neurite formation and the proliferation of NSCs during differentiation were enhanced by GaN with UV irradiation. Finally, the results of the Kelvin probe force microscope (KPFM) experiments showed that the NSCs cultured on GaN with UV irradiation displayed about 50 mV higher potential than those cultured on GaN without irradiation. The increase in cell membrane potential may have been due to the larger number of photogenerated free charges on the GaN surface with UV irradiation. These results could benefit topical research and the application of GaN as a biomedical material integrated into neural interface systems or other bioelectronic devices.

Selective degradation of mutant FMS-like tyrosine kinase-3 requires BIM-dependent depletion of heat shock proteins

AbstractInternal tandem duplications in the FMS-like tyrosine kinase-3 (FLT3-ITD) are common mutations in acute myeloid leukemia (AML). Proteolysis-targeting chimeras (PROTACs) that induce proteasomal degradation of mutated FLT3 emerge as innovative pharmacological approach. Molecular mechanisms that control targeted proteolysis beyond the ubiquitin-proteasome-system are undefined and PROTACs are the only known type of FLT3 degraders. We report that the von-Hippel-Lindau ubiquitin-ligase based FLT3 PROTAC MA49 (melotinib-49) and the FLT3 hydrophobic tagging molecule MA50 (halotinib-50) reduce endoplasmic reticulum-associated, oncogenic FLT3-ITD but spare FLT3. Nanomolar doses of MA49 and MA50 induce apoptosis of human leukemic cell lines and primary AML blasts with FLT3-ITD (p < 0.05-0.0001), but not of primary hematopoietic stem cells and differentiated immune cells, FLT3 wild-type cells, retinal cells, and c-KIT-dependent cells. In vivo activity of MA49 against FLT3-ITD-positive leukemia cells is verified in a Danio rerio model. The degrader-induced loss of FLT3-ITD involves the pro-apoptotic BH3-only protein BIM and a previously unidentified degrader-induced depletion of protein-folding chaperones. The expression levels of HSP90 and HSP110 correlate with reduced AML patient survival (p < 0.1) and HSP90, HSP110, and BIM are linked to the expression of FLT3 in primary AML cells (p < 0.01). HSP90 suppresses degrader-induced FLT3-ITD elimination and thereby establishes a mechanistically defined feed-back circuit.

138 Regulation of post protein translational modification in adipogenesis and fat accumulation in pigs

Abstract In this study, we explored the role of protein transcriptional modification (PTM) in the adipogenesis and fat accumulation of intramuscular adipocytes. Intramuscular fat (IMF) is mainly stored in adipocytes interspersed in the perimysial space or within fascicles, and its content is a key factor attributing to meat quality in farm animals, especially in terms of tenderness, flavor and juiciness. The early stage from the fetus to the neonate is crucial for skeletal muscle development involving myogenesis, adipogenesis, and fibrogenesis, and is susceptible to environmental factors. Previous studies have been subjected to explore the molecular mechanism involved in IMF development. However, it was obscured by muscular background of IMF adipocytes. Therefore, we investigated PTM dynamics from three aspects: 1) first analyzed PTM levels of lysine acetylation (Kac), 2-hydroxyisobutyrylation (Khib), crotonylation (Kcr) and succinylation (Ksu) in three distinct differentiation potenctial primary stem cells, including myogenic precursor cells (MPCs), fibro-adipogenic progenitors (FAPs) and MSCs isolated from neonatal longissimus dorsi muscle of pigs within 3-d of age; 2) Comparing the Khib levels of proliferative and differentiated FAPs; and 3) Exploring the role of Khib modification in the adipose tissue of obese- and lean-types of pigs. We found that only Khib level in FAPs was significantly greater than MSCs. During adipogenic differentiation of FAPs, the modification level of Khib was significantly increased quadratically in parallel with protein levels of acylation regulatory enzymes KAT5 (writer) and HDAC2 (Eraser). In comparison of Khib levels in FAPs in proliferation and in differentiation and Khib levels in backfat tissues from obese pigs (Laiwu pigs) and lean pigs (Duroc pigs), we also demonstrated that Khib modification level was positive correlated with adipogenic differentiation and fat accumulation. Accordingly, the expression patterns of KAT5 and HDAC2 were parallel to the degree of lipid accumulation in the backfat adipose tissue of finishing pigs. Furthermore, we showed that KAT5 knockdown in FAPs inhibited adipogenic differentiation, while HDAC2 knockdown enhanced adipogenic differentiation of FAPs. By employing C3H10T1/2 cells, we identified EON1 involved in Khib modification in regulation of adipo gesis and fat accumulation with analysis of Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the Tandem Mass Tag (TMT Through conditional loss- and gain-of-function mutations, we further demonstrated that khib modification of ENO1 is necessary for preadipocyte differentiation and subsequent fat accumulation by mediating cellular glycolysis. In conclusion, we are the first to reveal the role of Khib modification in adipogenesis and fat deposition in pigs, and provided new clues for the improvement of fat accumulation and distribution via genetic selection and nutritional strategy in pigs.

Anti-aging Metabolite-Based Polymeric Microparticles for Intracellular Drug Delivery and Bone Regeneration.

Alpha-ketoglutarate (AKG), a key component of the tricarboxylic acid (TCA) cycle, has attracted attention for its anti-aging properties. Our recent study indicates that locally delivered cell-permeable AKG significantly promotes osteogenic differentiation and mouse bone regeneration. However, the cytotoxicity and rapid hydrolysis of the metabolite limit its application. In this study, we synthesize novel AKG-based polymeric microparticles (PAKG MPs) for sustained release. In vitro data suggest that the chemical components, hydrophilicity, and size of the MPs can significantly affect their cytotoxicity and pro-osteogenic activity. Excitingly, these biodegradable PAKG MPs are highly phagocytosable for nonphagocytic pre-osteoblasts MC3T3-E1 and primary bone marrow mesenchymal stem cells (BMSCs), significantly promoting their osteoblastic differentiation. RNAseq data suggest that PAKG MPs strongly activate Wnt/β-catenin and PI3K-Akt pathways for osteogenic differentiation. Moreover, PAKG enables poly (L-lactic acid) and poly (lactic-co-glycolic acid) MPs (PLLA & PLGA MPs) for efficient phagocytosis. Our data indicate that PLGA-PAKG MPs-mediated intracellular drug delivery can significantly promote stronger osteoblastic differentiation compared to PLGA MPs-delivered phenamil. Notably, PAKG MPs significantly improve large bone regeneration in a mouse cranial bone defect model. Thus, the novel PAKG-based MPs show great promise to improve osteogenic differentiation, bone regeneration, and enable efficient intracellular drug delivery for broad regenerative medicine.

Genome Engineering of Primary and Pluripotent Stem Cell-Derived Hepatocytes for Modeling Liver Tumor Formation.

Genome editing has demonstrated its utility in generating isogenic cell-based disease models, enabling the precise introduction of genetic alterations into wild-type cells to mimic disease phenotypes and explore underlying mechanisms. However, its application in liver-related diseases has been limited by challenges in genetic modification of mature hepatocytes in a dish. Here, we conducted a systematic comparison of various methods for primary hepatocyte culture and gene delivery to achieve robust genome editing of hepatocytes ex vivo. Our efforts yielded editing efficiencies of up to 80% in primary murine hepatocytes cultured in monolayer and 20% in organoids. To model human hepatic tumorigenesis, we utilized hepatocytes differentiated from human pluripotent stem cells (hPSCs) as an alternative human hepatocyte source. We developed a series of cellular models by introducing various single or combined oncogenic alterations into hPSC-derived hepatocytes. Our findings demonstrated that distinct mutational patterns led to phenotypic variances, affecting both overgrowth and transcriptional profiles. Notably, we discovered that the PI3KCA E542K mutant, whether alone or in combination with exogenous c-MYC, significantly impaired hepatocyte functions and facilitated cancer metabolic reprogramming, highlighting the critical roles of these frequently mutated genes in driving liver neoplasia. In conclusion, our study demonstrates genome-engineered hepatocytes as valuable cellular models of hepatocarcinoma, providing insights into early tumorigenesis mechanisms.

Epigenetic therapy potentiates transposable element transcription to create tumor-enriched antigens in glioblastoma cells.

Inhibiting epigenetic modulators can transcriptionally reactivate transposable elements (TEs). These TE transcripts often generate unique peptides that can serve as immunogenic antigens for immunotherapy. Here, we ask whether TEs activated by epigenetic therapy could appreciably increase the antigen repertoire in glioblastoma, an aggressive brain cancer with low mutation and neoantigen burden. We treated patient-derived primary glioblastoma stem cell lines, an astrocyte cell line and primary fibroblast cell lines with epigenetic drugs, and identified treatment-induced, TE-derived transcripts that are preferentially expressed in cancer cells. We verified that these transcripts could produce human leukocyte antigen class I-presented antigens using liquid chromatography with tandem mass spectrometry pulldown experiments. Importantly, many TEs were also transcribed, even in proliferating nontumor cell lines, after epigenetic therapy, which suggests that targeted strategies like CRISPR-mediated activation could minimize potential side effects of activating unwanted genomic regions. The results highlight both the need for caution and the promise of future translational efforts in harnessing treatment-induced TE-derived antigens for targeted immunotherapy.

DNA contamination within recombinant adeno-associated virus preparations correlates with decreased CD34+ cell clonogenic potential

Recombinant adeno-associated viruses (rAAV) are promising for applications in many genome editing techniques through their effectiveness as carriers of DNA homologous donors into primary hematopoietic stem and progenitor cells (HSPC) but have many outstanding concerns. Specifically, their biomanufacturing and the variety of factors that influence the quality and consistency of rAAV preps are in question. During the process of rAAV packaging, a cell line is transfected with several DNA plasmids that collectively encode all the necessary information to allow for viral packaging. Ideally, this process results in packaging of complete viral particles only containing rAAV genomes; however, this is not the case. Through this study, we were able to leverage Single-Stranded Virus (SSV)-seq, an NGS-based method to quantify all DNA species present within rAAV preps. From this, it was determined that much of the DNA within some rAAV preps is not vector-genome derived, and there is wide variability in the contamination by DNA across various preps. Furthermore, we demonstrate that transducing CD34+ hematopoietic stem and progenitor cells (HSPC) with preps with higher contaminating DNA resulted in decreased clonogenic potential, altered transcriptomic profiles, and decreased genomic editing. Collectively, this study characterized the effects of DNA contamination within rAAV preps on CD34+ HSPC cellular potential.

Breast Cancer Stem Cells Upregulate IRF6 in Stromal Fibroblasts to Induce Stromagenesis.

The microenvironment of a cancer stem cell (CSC) niche is often found in coexistence with cancer-associated fibroblasts (CAFs). Here, we show the first in-depth analysis of the interaction between primary triple-negative breast cancer stem cells (BCSCs) with fibroblasts. Using 2D co-culture models with specific seeding ratios, we identified stromal fibroblast aggregation at the BCSC cluster periphery, and, on closer observation, the aggregated fibroblasts was found to encircle BCSC clusters in nematic organization. In addition, collagen type I and fibronectin accumulation were also found at the BCSC-stromal periphery. MACE-Seq analysis of BCSC-encapsulating fibroblasts displayed the transformation of stromal fibroblasts to CAFs and the upregulation of fibrosis regulating genes of which the Interferon Regulatory Factor 6 (IRF6) gene was identified. Loss of function experiments with the IRF6 gene decreased fibroblast encapsulation around BCSC clusters in 2D co-cultures. In BCSC xenografts, fibroblast IRF6 expression led to an increase in the stromal area and fibroblast density in tumors, in addition to a reduction in necrotic growth. Based on our findings, we propose that fibroblast IRF6 function is an important factor in the development of the stromal microenvironment and in sustaining the BCSC tumor niche.

Bovine Placentome-Derived Extracellular Matrix: A Sustainable 3D Scaffold for Cultivated Meat.

Cultivated meat, an advancement in cellular agriculture, holds promise in addressing environmental, ethical, and health challenges associated with traditional meat production. Utilizing tissue engineering principles, cultivated meat production employs biomaterials and technologies to create cell-based structures by introducing cells into a biocompatible scaffold, mimicking tissue organization. Among the cell sources used for producing muscle-like tissue for cultivated meats, primary adult stem cells like muscle satellite cells exhibit robust capabilities for proliferation and differentiation into myocytes, presenting a promising avenue for cultivated meat production. Evolutionarily optimized for growth in a 3D microenvironment, these cells benefit from the biochemical and biophysical cues provided by the extracellular matrix (ECM), regulating cell organization, interactions, and behavior. While plant protein-based scaffolds have been explored for their utilization for cultivated meat, they lack the biological cues for animal cells unless functionalized. Conversely, a decellularized bovine placental tissue ECM, processed from discarded birth tissue, achieves the biological functionalities of animal tissue ECM without harming animals. In this study, collagen and total ECM were prepared from decellularized bovine placental tissues. The collagen content was determined to be approximately 70% and 40% in isolated collagen and ECM, respectively. The resulting porous scaffolds, crosslinked through a dehydrothermal (DHT) crosslinking method without chemical crosslinking agents, supported the growth of bovine myoblasts. ECM scaffolds exhibited superior compatibility and stability compared to collagen scaffolds. In an attempt to make cultivate meat constructs, bovine myoblasts were cultured in steak-shaped ECM scaffolds for about 50 days. The resulting construct not only resembled muscle tissues but also displayed high cellularity with indications of myogenic differentiation. Furthermore, the meat constructs were cookable and able to sustain the grilling/frying. Our study is the first to utilize a unique bovine placentome-derived ECM scaffold to create a muscle tissue-like meat construct, demonstrating a promising and sustainable option for cultivated meat production.

Stem cells tightly regulate dead cell clearance to maintain tissue fitness

Billions of cells are eliminated daily from our bodies1–4. Although macrophages and dendritic cells are dedicated to migrating and engulfing dying cells and debris, many epithelial and mesenchymal tissue cells can digest nearby apoptotic corpses1–4. How these non-motile, non-professional phagocytes sense and eliminate dying cells while maintaining their normal tissue functions is unclear. Here we explore the mechanisms that underlie their multifunctionality by exploiting the cyclical bouts of tissue regeneration and degeneration during hair cycling. We show that hair follicle stem cells transiently unleash phagocytosis at the correct time and place through local molecular triggers that depend on both lipids released by neighbouring apoptotic corpses and retinoids released by healthy counterparts. We trace the heart of this dual ligand requirement to RARγ–RXRα, whose activation enables tight regulation of apoptotic cell clearance genes and provides an effective, tunable mechanism to offset phagocytic duties against the primary stem cell function of preserving tissue integrity during homeostasis. Finally, we provide functional evidence that hair follicle stem cell-mediated phagocytosis is not simply redundant with professional phagocytes but rather has clear benefits to tissue fitness. Our findings have broad implications for other non-motile tissue stem or progenitor cells that encounter cell death in an immune-privileged niche.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in, and base editing. PERC involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating the formulation with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) bearing homology-directed repair template DNA may be included. In contrast to electroporation, PERC is appealing as it requires no dedicated hardware and has less impact on cell phenotype and viability. Due to the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Here we report methods for improved PERC-mediated editing of T cells as well as novel methods for PERC-mediated editing of HSPCs, including knockout and precise knock-in. Editing efficiencies can surpass 90% using either Cas9 or Cas12a in primary T cells or HSPCs. Because PERC calls for only three readily available reagents - protein, RNA, and peptide - and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes this approach appealing for rapid deployment in research and clinical settings.

7,8-DHF inhibits BMSC oxidative stress via the TRKB/PI3K/AKT/NRF2 pathway to improve symptoms of postmenopausal osteoporosis

Postmenopausal osteoporosis (PMO) is characterized by bone loss and microstructural damage, and it is most common in older adult women. Currently, there is no cure for PMO. The flavonoid chemical 7,8-dihydroxyflavone (7,8-DHF) specifically activates tropomyosin receptor kinase B (TRKB). Furthermore, 7,8-DHF has various biological characteristics, including anti-inflammatory and antioxidant effects. However, the specific implications and fundamental mechanisms of 7,8-DHF in PMO remain unclear. We used protein imprinting, flow cytometry, tissue staining, and other methods to estimate the preventive mechanisms of 7,8-DHF against hydrogen peroxide (H2O2)-induced apoptosis in primary mouse bone marrow mesenchymal stem cells (BMSCs), osteogenic differentiation ability, and bone mass in ovariectomized (OVX) mice. We found that 7,8-DHF effectively prevented H2O2-induced reductions in the viability and osteogenic differentiation capacity of primary BMSCs. Mechanistically, 7,8-DHF induced the TRKB to activate the PI3K/AKT/NRF2 pathway. In vivo experiments with the OVX mouse model confirmed that 7,8-DHF can inhibit oxidative stress and promote bone formation, indicating that 7,8-DHF improves the viability and osteogenic differentiation ability of BMSCs stimulated via H2O2 by activating the TRKB/PI3K/AKT and NRF2 pathways, thereby improving PMO.

Advances in manufacturing chimeric antigen receptor immune cell therapies.

Biomedical research has witnessed significant strides in manufacturing chimeric antigen receptor T cell (CAR-T) therapies, marking a transformative era in cellular immunotherapy. Nevertheless, existing manufacturing methods for autologous cell therapies still pose several challenges related to cost, immune cell source, safety risks, and scalability. These challenges have motivated recent efforts to optimize process development and manufacturing for cell therapies using automated closed-system bioreactors and models created using artificial intelligence. Simultaneously, non-viral gene transfer methods like mRNA, CRISPR genome editing, and transposons are being applied to engineer T cells and other immune cells like macrophages and natural killer cells. Alternative sources of primary immune cells and stem cells are being developed to generate universal, allogeneic therapies, signaling a shift away from the current autologous paradigm. These multifaceted innovations in manufacturing underscore a collective effort to propel this therapeutic approach toward broader clinical adoption and improved patient outcomes in the evolving landscape of cancer treatment. Here, we review current CAR immune cell manufacturing strategies and highlight recent advancements in cell therapy scale-up, automation, process development, and engineering.

CRISPR Dependency Screens in Primary Hematopoietic Stem Cells Identify KDM3B as a Genotype-specific Vulnerability in IDH2- and TET2-mutant Cells.

Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs. Significance: Given the broad prevalence, comorbidities, and risk of malignant transformation associated with CH, there is an unmet need to identify therapeutic targets. We develop an ex vivo platform to perform CRISPR/Cas9 screens in primary HSPCs. We identify KDM3B and downstream signaling components as genotype-specific dependencies in CH and myeloid malignancies. See related commentary by Khabusheva and Goodell, p. 1768.

Nobiletin restores HFD-induced enteric nerve injury by regulating enteric glial activation and the GDNF/AKT/FOXO3a/P21 pathway

BackgroundTo explore whether nobiletin has a protective effect on high-fat diet (HFD)-induced enteric nerve injury and its underlying mechanism.MethodsAn obesity model was induced by a HFD. Nobiletin (100 mg/kg and 200 mg/kg) and vehicle were administered by gastric gavage for 4 weeks. Lee’s index, body weight, OGTT and intestinal propulsion assays were performed before sacrifice. After sampling, lipids were detected using Bodipy 493/503; lipid peroxidation was detected using MDA and SOD kits and the expression of PGP 9.5, Trem2, GFAP, β-tubulin 3, Bax, Bcl2, Nestin, P75 NTR, SOX10 and EDU was detected using immunofluorescence. The GDNF, p-AKT, AKT, p-FOXO3a, FOXO3a and P21 proteins were detected using western blotting. The relative mRNA expression levels of NOS2 were detected via qPCR. Primary enteric neural stem cells (ENSCs) were cultured. After ENSCs were treated with palmitic acid (PA) and nobiletin, CCK-8 and caspase-3/7 activity assays were performed to evaluate proliferation and apoptosis.ResultsHFD consumption caused colon lipid accumulation and peroxidation, induced enteric nerve damage and caused intestinal motor dysfunction. However, nobiletin reduced lipid accumulation and peroxidation in the colon; promoted Trem2, β-tubulin 3, Nestin, P75NTR, SOX10 and Bcl2 expression; inhibited Bax and GFAP expression; reduced NOS2 mRNA transcription; and regulated the GDNF/AKT/FOXO3a/P21 pathway. Nobiletin also promoted PA-induced impairment of ENSCs.ConclusionsNobiletin restored HFD-induced enteric nerve injury, which may be associated with inhibiting enteric nerve apoptosis, promoting enteric nerve survival and regulating the GDNF/AKT/FOXO3a/P21 pathway.

Exploring the relationship between pyroptosis and inflammatory bone loss: Evidence from a cigarette smoke-induced osteoporosis mouse model

Smoking is by far one of the greatest public health threats and is recognized as an important predisposing factor for osteoporosis. Exposure to cigarette smoke (CS) has been reported to be associated with inflammation-associated diseases through the induction of pyroptosis. Nevertheless, the correlation between pyroptosis and bone loss induced by CS remains uninvestigated. Here, a mouse model of mainstream smoke exposure-induced osteoporosis was established. μCT, biomechanical testing, and immunohistochemical staining of bone tissue were used to assess the deleterious effects of CS on bone metabolism. In vitro, the effects of cigarette smoke extracts (CSE) on mouse primary bone marrow-derived mesenchymal stem cells (BMSCs) were tested by cell viability assays, gene and protein expression assays, and alizarin red staining. The utilization of the pyroptosis inhibitor MCC950 served to confirm the critical role of BMSCs pyroptosis in CS-induced osteoporosis. Our results indicated that exposure to mainstream smoke led to a notable decrease in the quantity of osteoblasts and hindered the process of osteogenic differentiation in mice. Additionally, there was a significant increase in the expression of pyroptosis-related proteins in the bone marrow. The inhibitory effects of CSE on cell viability and osteogenic differentiation of BMSCs were found to be dose-dependent in vitro. However, the presence of the pyroptosis inhibitor MCC950 significantly improved the impaired osteogenic differentiation and bone mineralization caused by CSE. These results highlight the crucial involvement of BMSCs pyroptosis in the development of bone loss induced by CS. In summary, the findings of this study provide novel evidence that CS exerts a detrimental effect on the process of osteogenesis in BMSCs through the induction of pyroptosis, ultimately leading to bone loss. Inhibition of pyroptosis effectively attenuated the toxicological effects of CS on BMSCs, providing a new target for preventing inflammatory osteoporosis.

Developing a framework for integrating STEM approach at primary education of Bangladesh

STEM (Science, Mathematics, Engineering and Technology) education has widely been considered to have the potential to prepare students with 21st-century skills. Though the Government of Bangladesh (GoB) is concerned with developing human resources for the 21st-century, STEM education has not yet achieved a strong position in the national curriculum system. This study aims to provide a guideline to the GoB to take necessary steps to integrate STEM initiatives aligned with the current science and mathematics curriculum. Adopting a literature survey on the integrated STEM approach, document analysis (primary math and science curriculum, textbook, and teachers’ guide of grade 3, 4, and 5) and semi-structured interviews of the primary mathematics teachers, head teachers, STEM experts and curriculum developers of Bangladesh, a framework of integrated STEM approach for primary level has been developed. Besides, the study identified some chapters from science and mathematics textbooks where there are some scopes to integrate the STEM approach and also developed some STEM activities for grade 3, 4, and 5 as future guidelines to the practitioners. This study contributes to the field of STEM-focused primary education in the context of Bangladesh by offering practical guidelines for academic institutions, curriculum developers, teachers’ trainers, and policy makers of the country.

The Leukemic Isocitrate Dehydrogenase (IDH) 1/2 Mutations Impair Myeloid and Erythroid Cell Differentiation of Primary Human Hematopoietic Stem and Progenitor Cells (HSPCs).

How hematopoietic stem and progenitor cell (HSPC) fate decisions are affected by genetic alterations acquired during AML leukemogenesis is poorly understood and mainly explored in animal models. Here, we study isocitrate dehydrogenase (IDH) gene mutations in the human model of HSPC and discuss the available literature on this topic. IDH1/2 mutations occur in ~20% of AML cases, are recognized among the mutations earliest acquired during leukemogenesis, and are targets of specific inhibitors (ivosidenib and enasidenib, respectively). In order to investigate the direct effects of these mutations on HSPCs, we expressed IDH1-R132H or IDH2-R140Q mutants into human CD34+ healthy donor cells via lentiviral transduction and analyzed the colony-forming unit (CFU) ability. CFU ability was dramatically compromised with a complete trilineage block of differentiation. Strikingly, the block was reversed by specific inhibitors, confirming that it was a specific effect induced by the mutants. In line with this observation, the CD34+ leukemic precursors isolated from a patient with IDH2-mutated AML at baseline and during enasidenib treatment showed progressive and marked improvements in their fitness over time, in terms of CFU ability and propensity to differentiate. They attained clonal trilinear reconstitution of hematopoiesis and complete hematological remission.

Accelerating Diverse Cell-Based Therapies Through Scalable Design.

Augmenting cells with novel, genetically encoded functions will support therapies that expand beyond natural capacity for immune surveillance and tissue regeneration. However, engineering cells at scale with transgenic cargoes remains a challenge in realizing the potential of cell-based therapies. In this review, we introduce a range of applications for engineering primary cells and stem cells for cell-based therapies. We highlight tools and advances that have launched mammalian cell engineering from bioproduction to precision editing of therapeutically relevant cells. Additionally, we examine how transgenesis methods and genetic cargo designs can be tailored for performance. Altogether, we offer a vision for accelerating the translation of innovative cell-based therapies by harnessing diverse cell types, integrating the expanding array of synthetic biology tools, and building cellular tools through advanced genome writing techniques.

The Principle of Cortical Development and Evolution.

Human's robust cognitive abilities, including creativity and language, are made possible, at least in large part, by evolutionary changes made to the cerebral cortex. This paper reviews the biology and evolution of mammalian cortical radial glial cells (primary neural stem cells) and introduces the concept that a genetically step wise process, based on a core molecular pathway already in use, is the evolutionary process that has molded cortical neurogenesis. The core mechanism, which has been identified in our recent studies, is the extracellular signal-regulated kinase (ERK)-bone morphogenic protein 7 (BMP7)-GLI3 repressor form (GLI3R)-sonic hedgehog (SHH) positive feedback loop. Additionally, I propose that the molecular basis for cortical evolutionary dwarfism, exemplified by the lissencephalic mouse which originated from a larger gyrencephalic ancestor, is an increase in SHH signaling in radial glia, that antagonizes ERK-BMP7 signaling. Finally, I propose that: (1) SHH signaling is not a key regulator of primate cortical expansion and folding; (2) human cortical radial glial cells do not generate neocortical interneurons; (3) human-specific genes may not be essential for most cortical expansion. I hope this review assists colleagues in the field, guiding research to address gaps in our understanding of cortical development and evolution.