Cell Differentiation Research Articles

Akkermansia muciniphila identified as key strain to alleviate gut barrier injury through Wnt signaling pathway.

As the largest mucosal surface, the gut has built a physical, chemical, microbial, and immune barrier to protect the body against pathogen invasion. The disturbance of gut microbiota aggravates pathogenic bacteria invasion and gut barrier injury. Fecal microbiota transplantation (FMT) is a promising treatment for microbiome-related disorders, where beneficial strain engraftment is a significant factor influencing FMT outcomes. The aim of this research was to explore the effect of FMT on antibiotic-induced microbiome-disordered (AIMD) models infected with enterotoxigenic Escherichia coli (ETEC). We used piglet, mouse, and intestinal organoid models to explore the protective effects and mechanisms of FMT on ETEC infection. The results showed that FMT regulated gut microbiota and enhanced the protection of AIMD piglets against ETEC K88 challenge, as demonstrated by reduced intestinal pathogen colonization and alleviated gut barrier injury. Akkermansia muciniphila (A. muciniphila) and Bacteroides fragilis (B. fragilis) were identified as two strains that may play key roles in FMT. We further investigated the alleviatory effects of these two strains on ETEC infection in the AIMD mice model, which revealed that A. muciniphila and B. fragilis relieved ETEC-induced intestinal inflammation by maintaining the proportion of Treg/Th17 cells and epithelial damage by moderately activating the Wnt/β-catenin signaling pathway, while the effect of A. muciniphila was better than B. fragilis. We, therefore, identified whether A. muciniphila protected against ETEC infection using basal-out and apical-out intestinal organoid models. A. muciniphila did protect the intestinal stem cells and stimulate the proliferation and differentiation of intestinal epithelium, and the protective effects of A. muciniphila were reversed by Wnt inhibitor. FMT alleviated ETEC-induced gut barrier injury and intestinal inflammation in the AIMD model. A. muciniphila was identified as a key strain in FMT to promote the proliferation and differentiation of intestinal stem cells by mediating the Wnt/β-catenin signaling pathway.

Akkermansia muciniphila identified as key strain to alleviate gut barrier injury through Wnt signaling pathway

As the largest mucosal surface, the gut has built a physical, chemical, microbial, and immune barrier to protect the body against pathogen invasion. The disturbance of gut microbiota aggravates pathogenic bacteria invasion and gut barrier injury. Fecal microbiota transplantation (FMT) is a promising treatment for microbiome-related disorders, where beneficial strain engraftment is a significant factor influencing FMT outcomes. The aim of this research was to explore the effect of FMT on antibiotic-induced microbiome-disordered (AIMD) models infected with enterotoxigenic Escherichia coli (ETEC). We used piglet, mouse, and intestinal organoid models to explore the protective effects and mechanisms of FMT on ETEC infection. The results showed that FMT regulated gut microbiota and enhanced the protection of AIMD piglets against ETEC K88 challenge, as demonstrated by reduced intestinal pathogen colonization and alleviated gut barrier injury. Akkermansia muciniphila (A. muciniphila) and Bacteroides fragilis (B. fragilis) were identified as two strains that may play key roles in FMT. We further investigated the alleviatory effects of these two strains on ETEC infection in the AIMD mice model, which revealed that A. muciniphila and B. fragilis relieved ETEC-induced intestinal inflammation by maintaining the proportion of Treg/Th17 cells and epithelial damage by moderately activating the Wnt/β-catenin signaling pathway, while the effect of A. muciniphila was better than B. fragilis. We, therefore, identified whether A. muciniphila protected against ETEC infection using basal-out and apical-out intestinal organoid models. A. muciniphila did protect the intestinal stem cells and stimulate the proliferation and differentiation of intestinal epithelium, and the protective effects of A. muciniphila were reversed by Wnt inhibitor. FMT alleviated ETEC-induced gut barrier injury and intestinal inflammation in the AIMD model. A. muciniphila was identified as a key strain in FMT to promote the proliferation and differentiation of intestinal stem cells by mediating the Wnt/β-catenin signaling pathway.

Spatially controlled multicellular differentiation of stem cells using triple factor-releasing metal–organic framework-coated nanoline arrays

Improved in vitro models are needed for regenerative therapy and drug screening. Here, we report on functionally aligned nanoparticle-trapped nanopattern arrays for spatially controlled, precise mesenchymal stem cell differentiation on a single substrate. The arrays comprise nanohole and nanoline arrays fabricated through interference lithography and selectively capture of UiO-67 metal–organic frameworks on nanoline arrays with a 99.8% efficiency using an optimised asymmetric spin-coating method. The UiO-67 metal–organic frameworks contain three osteogenic differentiation factors for sustained release over four weeks. The combination of differentiation factors and patterned array allows for generation of adipocytes, osteoblasts, and adipocyte–osteoblast mixtures on nanohole arrays, nanoline arrays, and at the nanohole–nanoline interface, respectively, with mature osteoblasts exhibiting higher marker expression and mineralisation. The sustained release patterned array holds potential for constructing advanced therapeutic and disease state in vitro cellular models.

Vitamin D Enhancement of Adipose Biology: Implications on Obesity-Associated Cardiometabolic Diseases

Vitamin D is activated into 1α,25(OH)2D through two hydroxylation steps that are primarily catalyzed by 25-hydroxylase in the liver and 1α-hydroxylase in the kidneys. The active form of vitamin D regulates myriads of cellular functions through its nuclear receptor, vitamin D receptor (VDR). Vitamin D metabolizing enzymes and VDR are expressed in adipose tissues and vitamin D regulates multiple aspects of adipose biology including the recruitment and differentiation of adipose stem cells into adipocytes and metabolic, endocrine, and immune properties. Obesity is associated with low vitamin D status, which is thought to be explained by its sequestration in large mass of adipose tissues as well as dysregulated vitamin D metabolism. Low vitamin D status in obesity may negatively impact adipose biology leading to adipose tissue dysfunctions, the major pathological factors for cardiometabolic diseases in obesity. In this review, the current understanding of vitamin D metabolism and its molecular mechanisms of actions, focusing on vitamin D–VDR regulation of adipose biology with their implications on obesity-associated diseases, is discussed. Whether improving vitamin D status leads to reductions in adiposity and risks for cardiometabolic diseases is also discussed.

Adsorption of Serum Fetuin onto Octacalcium Phosphate and Its Relation to Osteogenic Property

This study aimed to investigate how the chemical elements in relation to octacalcium phosphate (OCP) hydrolysis affect the osteoblastic differentiation in the presence of serum fetuin. The adsorption of fetuin onto OCP was examined in buffers having different degrees of supersaturation (DS) with respect to OCP and hydroxyapatite (HA) at pH 7.4 and 37 °C. The osteoblastic differentiation of mesenchymal stem cells (MSCs) was evaluated in cultures with OCP and 0 to 0.8 mg/mL of fetuin. The amount of fetuin adsorbed increased with increasing DS in the buffer. In the MSC culture, the coexistence of OCP and 0.2–0.4 mg/mL of fetuin close to serum level increased alkaline phosphatase activity; however, the activity was suppressed by 0.2–0.8 mg/mL of fetuin. Transmission electron microscopy revealed de novo crystal formation on OCP in supersaturated buffer and culture media with respect to OCP and HA at lower fetuin concentrations. Infrared spectroscopy and DS estimation indicate that the hydrolysis of OCP with de novo apatite formation was promoted in the culture media at 0.2–0.4 mg/mL of fetuin. These results suggest that OCP may promote osteoblastic differentiation if the suitable conditions are attained regarding the chemical elements and fetuin adsorption around OCP.

Development of 3D Muscle Cell Culture-Based Screening System for Metabolic Syndrome Drug Research.

Developing effective drug screening methods for type 2 diabetes requires physiologically relevant models. Traditional 2D cell cultures have limitations in replicating invivo conditions, leading to challenges in assessing drug efficacy. To overcome these issues, we developed a 3D artificial muscle model that induces insulin resistance, a hallmark of type 2 diabetes. Using C2C12 myoblasts cultured in a scaffold of 1% alginate and 1 mg/mL collagen type 1, we optimized conditions for differentiation and structural stability. Insulin resistance was induced using palmitic acid (PA), and glucose uptake was assessed using the fluorescent glucose analog 2-NBDG. The 3D model demonstrated superior glucose uptake responses compared with 2D cultures, with a threefold increase in insulin-stimulated glucose uptake on days 4 and 8 of differentiation. Induced insulin resistance was observed with 0.1 mM PA, which maintained cell viability and differentiation capacity. The model was validated through comparative drug screening using rosiglitazone and metformin, as well as 165 candidate compounds provided by Korea Chemical Bank. Drug screening revealed that three out of five hit compounds identified in both 2D and 3D models exhibited greater efficacy in 3D cultures, with results consistent with ex vivo assays using mouse soleus muscle. This model closely mimics invivo conditions, offering a robust platform for type 2 diabetes drug discovery while supporting ethical research practices.

Expansion of induced pluripotent stem cells under consideration of bioengineering aspects: part 2.

The manufacturing of allogeneic cell therapeutics based on human-induced pluripotent stem cells (hiPSCs) holds considerable potential to revolutionize the accessibility and affordability of modern healthcare. However, achieving the cell yields necessary to ensure robust production hinges on identifying suitable and scalable single-use (SU) bioreactor systems. While specific stirred SU bioreactor types have demonstrated proficiency in supporting hiPSC expansion at L-scale, others, notably instrumented SU multiplate and fixed-bed bioreactors, remain relatively unexplored. By characterizing these bioreactors using both computational fluid dynamics and experimental bioengineering methods, operating ranges were identified for the Xpansion® 10 and Ascent™ 1m2 bioreactors in which satisfactory hiPSC expansion under serum-free conditions was achieved. These operating ranges were shown not only to effectively limit cell exposure to wall shear stress but also facilitated sufficient oxygen transfer and mixing. Through their application, almost 5 × 109 viable cells could be produced within 5days, achieving expansion factorsof up to 35 without discernable impact on cell viability, identity, or differentiation potential. Key Points •Bioengineering characterizations allowed the identification of operating ranges that supported satisfactory hiPSC expansion •Both the Xpansion® 10 multiplate and Ascent™ 1m2 fixed-bed reactor accommodated the production of almost 5 × 109 viable cells within 5 days •Exposing the hiPSCs to a median wall shear stress of up to 8.2 × 10-5 N cm-2did not impair quality.

PIWI proteins and piRNAs: key regulators of stem cell biology

In this mini review, we discussed the functional roles of PIWI proteins and their associated small RNAs, piRNAs, in regulating gene expression within stem cell biology. Guided by piRNAs, these proteins transcriptionally and post-transcriptionally repress transposons using mechanisms such as the ping-pong amplification cycle and phasing to protect germline genomes. Initially identified in Drosophila melanogaster, the piRNA pathway regulate germline stem cell self-renewal and differentiation via cell-autonomous and non-cell-autonomous mechanisms. Precisely, in GSCs, PIWI proteins and piRNAs regulate gene expression by modulating chromatin states and directly influencing mRNA translation. For instance, the PIWI protein Aubergine loaded with piRNAs promotes and represses translation of certain mRNAs to balance self-renewal and differentiation. Thus, the piRNA pathway exhibits dual regulatory roles in mRNA stability and translation, highlighting its context-dependent functions. Moreover, PIWI proteins are essential in somatic stem cells to support the regenerative capacity of highly regenerative species, such as planarians. Similarly, in Drosophila intestinal stem cells, the PIWI protein Piwi regulates metabolic pathways and genome integrity, impacting longevity and gut homeostasis. In this case, piRNAs appear absent in the gut, suggesting piRNA-independent regulatory mechanisms. Together, PIWI proteins and piRNAs demonstrate evolutionary conservation in stem cell regulation, integrating TE silencing and gene expression regulation at chromatin and mRNA levels in somatic and germline lineages. Beyond their canonical roles, emerging evidence reveal their broader significance in maintaining stem cell properties and organismal health under physiological and pathological conditions.

SMURF1-Induced Ubiquitination of FTH1 Disrupts Iron Homeostasis and Suppresses Myogenesis

Ferritin heavy chain 1 (FTH1) is pivotal in the storage, release, and utilization of iron, plays a crucial role in the ferroptosis pathway, and exerts significant impacts on various diseases. Iron influences skeletal muscle development and health by promoting cell growth, ensuring energy metabolism and ATP synthesis, maintaining oxygen supply, and facilitating protein synthesis. However, the precise molecular mechanisms underlying iron’s regulation of skeletal muscle growth and development remain elusive. In this study, we demonstrated that the conditional knockout (cKO) of FTH1 in skeletal muscle results in muscle atrophy and impaired exercise endurance. In vitro studies using FTH1 cKO myoblasts revealed notable decreases in GSH concentrations, elevated levels of lipid peroxidation, and the substantial accumulation of Fe2+, collectively implying the induction of ferroptosis. Mechanistically, E3 ubiquitin-protein ligase SMURF1 (SMURF1) acts as an E3 ubiquitin ligase for FTH1, thereby facilitating the ubiquitination and subsequent degradation of FTH1. Consequently, this activation of the ferroptosis pathway by SMURF1 impedes myoblast differentiation into myotubes. This study identifies FTH1 as a novel regulator of muscle cell differentiation and skeletal muscle development, implicating its potential significance in maintaining skeletal muscle health through the regulation of iron homeostasis.

The essential role of cerebrospinal fluid in the brain; a comprehensive review.

There has been a significant amount of attention directed towards understanding brain development, shedding light on the underlying mechanisms. The proliferation and differentiation of brain stem cells have been a key focus. The process of neurolation occurs during the early stages of embryonic development, leading to the formation of the neural tube, a hollow nerve cord that gives rise to the central nervous system (CNS). There is a growing emphasis on the fluid-filled space inside the developing CNS and the potential role of cerebrospinal fluid (CSF) in brain development. The flow of CSF near the germinal epithelium significantly impacts the proliferation of cells in the cerebral cortex. CSF provides crucial support to the germinal epithelium, influencing the growth and differentiation of neural stem cells. It achieves this by releasing growth factors, cytokines, and morphogens that control the proliferation, survival, and migration of neuroepithelium. During development, the concentration of proteins in the CSF is notably higher compared to that in adults. Studies have indicated that removing CSF from the brain's ventricles during development causes an increase in neural cell deaths and a reduction in neural cell proliferation, ultimately leading to a thinner cerebral cortex. Additionally, many researches demonstrate that the composition of the CSF is essential for maintaining germinal matrix function and output, highlighting the critical role of CSF in brain development. It is concluded that CSF impacts the proliferation and differentiation of neural stem cells, which in turn plays a pivotal role in brain development.

SiRNA-Mimetic Ratiometric pH (sMiRpH) Probes for Improving Cell Delivery and mRNA Knockdown.

Second-generation siRNA-mimetic ratiometric pH probes (sMiRpH-2) were developed by hybridizing a 3'-FAM-labeled 2'-OMe RNA strand with a 3'-Cy5-labeled 25mer RNA strand. These duplexes demonstrated the silencing of cytoplasmic mRNA targets in HeLa cells as measured by RT-qPCR and supported by western blot analysis. Fluorescence intensity and lifetime measurements revealed that a single guanosine (G) positioned adjacent to FAM achieves substantial static quenching at pH 5, with additional collisional quenching rendering the dye almost nonemissive. A FAM-G π-π stacking interaction was evidenced by a red-shifted absorbance spectrum for FAM. Decreased quenching at near-neutral pH enhances the FAM dynamic range in the physiologic pH window and improves the differentiation in cells between endocytic entrapment and cytoplasmic release. Flow cytometric analysis of intracellular pH and uptake using sMiRpH-2 was corroborated by live cell confocal microscopy and found to be predictive of knockdown efficacy. A sMiRpH-2 probe successfully predicted the relative efficacy of two transfection agents in more challenging SK-OV-3 cells, which highlights its use for the rapid assessment of nonviral siRNA delivery vectors.

Therapeutic Potential of Adina rubella Hance Stem and Picroside III as a Differentiation Inducer in AML Cells via Mitochondrial ROS Accumulation

Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells and a differentiation block, highlighting the urgent need for novel differentiation-inducing therapies. This study evaluated Adina rubella Hance (ARH) stem as a potent differentiation inducer by systematically screening 200 plant extracts. ARH stem promoted phenotypic differentiation in AML cells. In addition to its differentiation-inducing effects, ARH stem exhibited strong antileukemic activities, such as inhibiting cell proliferation, inducing cell death, and enhancing mitochondrial reactive oxygen species (mtROS) levels, the latter of which is critical for its differentiation-promoting activity. Comparative analysis with the extracts from other parts of the plant confirmed the superior efficacy of the stem extract because of its unique chemical composition. Ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry analysis identified Picroside III as a major active compound within the stem extract, capable of recapitulating ARH stem-induced differentiation and demonstrating significant antileukemic properties. These findings underscore the therapeutic potential of ARH stem and its active component, Picroside III, as promising agents for differentiation-based treatment strategies in AML.

Hollow‐Tube‐Whisker‐Modified Biphasic Calcium Phosphate Ceramics Loaded with SDF‐1α and EPCs for Steroid‐Induced Osteonecrosis of Femoral Head

AbstractSteroid‐induced osteonecrosis of the femoral head (SONFH) remains a significant challenge in orthopedic clinical treatment. In this study, a novel 3D hollow‐tube‐whisker‐modified biphasic calcium phosphate ceramic (H‐BCP) is successfully fabricated using an in‐situ growth technique. Compared to conventional BCP ceramics, H‐BCP exhibited increased specific surface area, enhanced mechanical strength, and improved biocompatibility. Utilizing the hollow‐tube whisker structure, H‐BCP is functionalized with stromal cell‐derived factor‐1α (SDF‐1α) to construct the H‐BCP@SDF‐1α sustained‐release system. In vitro studies demonstrated that H‐BCP@SDF‐1α effectively recruited bone marrow stromal cells (BMSCs), promoted their osteogenic differentiation, and supported the angiogenic differentiation of endothelial progenitor cells (EPCs). Furthermore, EPC‐loaded H‐BCP@SDF‐1α (H‐BCP@SDF‐1α/EPC) is used to construct vascularized tissue‐engineered bone and implanted into a SONFH rabbit model following core decompression surgery. At 12 weeks post‐surgery, animals implanted with H‐BCP@SDF‐1α/EPC exhibited significant new bone formation, bone integration, and in situ revascularization. Additionally, it is found that H‐BCP@SDF‐1α/EPC activated the PI3K/AKT signaling pathway in BMSCs, upregulating osteogenic gene expression. This study explores a novel material system integrating mechanical support, bioactivity, and drug delivery, offering a promising approach for SONFH treatment.

Mixed-valence vanadium-doped mesoporous bioactive glass for treatment of tumor-associated bone defects.

Vanadium is a bioactive trace element with variable valence. Its pentavalent form has been confirmed to be capable of predominantly regulating the early and mid-stage osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) without tumor inhibition, while its tetravalent form exhibits tumor inhibition but only primarily modulates late osteogenic differentiation and angiogenesis. In this study, a multifunctional bone tissue scaffold consisting of mixed-valence vanadium-doped mesoporous bioactive glass and poly(lactic-co-glycolic acid) (V(IV/V)-MBG/PLGA) was developed to simultaneously inhibit the recurrence of osteosarcoma and promote the regeneration of operative bone defects. The in vitro results showed that the V(IV) and V(V) species could be sustainably released from V(IV/V)-MBG and complementarily enhance the proliferation, osteogenic differentiation, and mineralization of BMSCs by activating multiple signaling pathways throughout the whole osteogenesis process. More importantly, the co-existence of mixed-valent vanadium species was able to continuously stimulate the generation of excessive ROS and the depletion of GSH by synergistically supplying an appropriate ratio of V(IV) and V(V) to thermodynamically and kinetically maintain the stable self-circulation of the valence state alteration, thus inducing UMR-106 cell death. In a rat model, V(IV/V)-MBG/PLGA scaffolds effectively suppressed tumor invasion and promoted bone regeneration. These results suggest that V(IV/V)-MBG/PLGA scaffolds are a promising strategy for treating tumor-associated bone defects, offering dual tumor inhibition and bone regeneration.

Rac1-dependent regulation of osteoclast and osteoblast differentiation by developmentally regulated GTP-binding 2

Multiple small GTPases play crucial roles in bone homeostasis by regulating the differentiation and function of bone cells, including osteoclasts and osteoblasts. Here, we investigated whether developmentally regulated GTP-binding protein 2 (Drg2), a subfamily of the GTPase superfamily, could affect bone mass by regulating osteoclast and osteoblast differentiation. Downregulation of Drg2 using siRNA in bone marrow-derived macrophages inhibited osteoclast differentiation and function and Rac1 activation in vitro. Comparatively, Drg2 downregulation in calvarial-derived osteoprogenitor cells enhanced osteoblast differentiation and function in vitro. Rac1 activation was also suppressed by Drg2 downregulation in osteoprogenitor cells. Both osteoclast and osteoblast differentiation regulated by Drg2 downregulation were restored by suppressing Rac1 activity. Drg2-deficient mice showed increased bone mass due to a dramatic reduction in osteoclast numbers without significantly affecting the number of osteoblasts. Furthermore, Drg2 downregulation strongly inhibited RANKL-induced bone loss in vivo. In summary, Drg2 contributes to bone homeostasis by regulating the differentiation and function of osteoclasts and osteoblasts through Rac1 activation. In particular, the effect of Drg2 on osteoclasts is strong enough to regulate bone mass in vivo; therefore, Drg2 has significant potential for use as a therapeutic target in bone loss-related diseases.

Chiral Supramolecular Hydrogels Regulating Both Osteoblastogenesis and Osteoclastogenesis

Osteoporosis, a chronic bone disorder, poses a global threat to the health of millions of individuals. The disruption of bone homeostasis is the fundamental cause of osteoporosis. Currently, clinical drugs are employed to promote bone formation via enhancing osteogenesis and/or reduce bone loss via inhibiting osteoclastogenesis. However, it is difficult for the current drugs to simultaneously address the osteoblastogenesis and osteoclastogenesis issues associated with osteoporosis. Hence, L/D-phenylalanine derivatives (L/DPF), combined with Mg2+ ions, are employed to assemble into chiral supramolecular hydrogels which facilitate osteocyte activity and inhibit osteoclast function. LPF_Mg hydrogels and DPF_Mg hydrogels demonstrate the opposite supramolecular chirality. Specifically, LPF_Mg hydrogels and DPF_Mg hydrogels are composed of left-handed (M-type) helical nanofibers and right-handed (P-type) helical nanofibers, respectively. The hydrogen bonding and π–π stacking interactions are crucial in the process of hydrogel formation. The chiral left-handed nanofibrous DPF_Mg hydrogels significantly promote osteogenic differentiation of MC3T3 cells and inhibit osteoclast differentiation of RAW267.4 cells, thereby demonstrating substantial potential for applications in improving skeletal health. These findings provide a promising novel perspective on the application of chiral functional materials for osteoporosis therapy.

Full-length mRNA sequencing resolves novel variation in 5' UTR length for genes expressed during human CD4 T-cell activation.

Isoform sequencing (Iso-Seq) uses long-read technology to produce highly accurate full-length reads of mRNA transcripts. Visualization of individual mRNA molecules can reveal new details of transcript variation within understudied portions of mRNA, such as the 5' untranslated region (UTR). Differential 5' UTRs may contain motifs, upstream open reading frames (uORFs), and secondary structures that can serve to regulate translation or further indicate changes in promoter usage, where transcriptional control may impact protein expression levels. To begin to explore isoform variation during T-cell activation, we generated the first Iso-Seq reference transcriptome of activated human CD4 T cells. Within this dataset, we discovered many novel splice- and end-variant transcripts. Remarkably, one in every eight genes expressed in our dataset was found to have a notable proportion of transcripts with 5' UTR lengthened by over 100bp compared to the longest corresponding UTR within the Gencode dataset. Among these end-variant transcripts, two novel isoforms were identified for CXCR5, a chemokine receptor associated with T follicular helper cell (Tfh) function and differentiation. When investigated in a model cell system, these lengthened UTR conferred reduced transcript stability and, for one of these isoforms, short uORFs introduced by the added length altered protein expression kinetics. This study highlights instances in which current reference databases are incomplete relative to the information obtained by long-read sequencing of intact mRNA. Iso-Seq is thus a promising approach to better understanding the plasticity of promoter usage, alternative splicing, and UTR sequences that influence RNA stability and translation efficiency.

The role of RAP2 in regulation of cell volume on bone marrow mesenchymal stem cell fate determination.

The extracellular matrix guides cell behavior through mechanical properties, which plays a role in determining cell function and can even influence stem cell fate. Compared with adherent culture, the three-dimensional culture environment is closer to the growth conditions in vivo, but is limited by standardization of material properties and observation and measurement methods. Therefore, it is necessary to study the relationship among the three-dimensional morphological characteristics of cells, cytoskeleton, and stem cell differentiation under adherent culture conditions. Here, we control the cell volume by adjusting the cell density, microfilament cytoskeleton tension, and osmotic pressure of the culture environment, and analyze the cell morphological features and differentiation to the osteoblastic and adipogenic lineages. Based on the in vitro and in vivo results, we identify cell volume as the true reflection of the cytoskeleton tension under stress stimuli compared with cell spreading area. By adjusting cell volume, cytoskeletal tension and cell differentiation can be regulated without affecting cell spreading area. Further study shows that the Ras-related small GTPase RAP2 inhibits the activity of mechanical transducers Lamin A/C and YAP1, playing an important role in cell volume regulation of cell differentiation. In summary, our results support the close relationship between cell volume and cytoskeleton tension. The regulatory role of cell volume on cell differentiation is modulated, at least in part, by RAP2-related mechanosensitive pathways. Our insights into how cell volume regulates cell differentiation may build a bridge between two-dimensional and three-dimensional mechanical studies in cell biology.

ROCK1 promotes B-cell differentiation and proteostasis under stress through the heme-regulated proteins, BACH2 and HRI.

The mechanisms utilized by differentiating B cells to withstand highly damaging conditions generated during severe infections, like the massive hemolysis that accompanies malaria, are poorly understood. Here we demonstrate that ROCK1 regulates B cells differentiation in hostile environments replete with PAMPs (pathogen-associated molecular patterns) and high levels of heme by controlling two key heme-regulated molecules, BACH2 and Heme-regulated eIF2a kinase (HRI). ROCK1 phosphorylates BACH2 and protects it from heme-driven degradation. As B cells differentiate, furthermore, ROCK1 restrains their proinflammatory potential and helps them handle the heightened stress imparted by the presence of PAMPs and heme by controlling HRI, a key regulator of the integrated stress response and cytosolic proteotoxicity. ROCK1 controls the interplay of HRI with HSP90 and limits the recruitment of HRI and HSP90 to unique p62/SQSTM1 complexes that also contain critical kinases like mTORC1 and TBK1, and proteins involved in RNA metabolism, oxidative damage, and proteostasis like TDP-43. Thus, ROCK1 helps B cells cope with intense pathogen-driven destruction by coordinating the activity of key controllers of B cell differentiation and stress responses. These ROCK1-dependent mechanisms may be widely employed by cells to handle severe environmental stresses, and these findings may be relevant for immune-mediated and age-related neurodegenerative disorders.

Cell–cell heterogeneity in phosphoenolpyruvate carboxylase biases early cell fate priming in Dictyostelium discoideum

Glucose metabolism is a key factor characterizing the cellular state during multicellular development. In metazoans, the metabolic state of undifferentiated cells correlates with growth/differentiation transition and cell fate determination. Notably, the cell fate of the Amoebozoa species Dictyostelium discoideum is biased by the presence of glucose and is also correlated with early differences in intracellular ATP. However, the relationship between early cell–cell heterogeneity, cell differentiation, and the metabolic state is unclear. To address the link between glucose metabolism and cell differentiation in D. discoideum, we studied the role of phosphoenolpyruvate carboxylase (PEPC), a key enzyme in the PEP-oxaloacetate-pyruvate node, a core junction that dictates the metabolic flux of glycolysis, the TCA cycle, and gluconeogenesis. We demonstrate that there is cell–cell heterogeneity in PEPC promoter activity in vegetative cells, which depends on nutrient conditions, and that cells with high PEPC promoter activity differentiate into spores. The PEPC null mutant exhibited an aberrantly high prestalk/prespore ratio, and the spore mass of the fruiting body was glassy and consisted of immature spores. Furthermore, the PEPC null mutant had high ATP levels and low mitochondrial membrane potential. Our results suggest the importance of cell–cell heterogeneity in the levels of metabolic enzymes during early cell fate priming.