Cell Fate Transitions Research Articles

Cell-type-specific loops linked to RNA polymerase II elongation in human neural differentiation

DNA is folded into higher-order structures that shape and are shaped by genome function. The role of long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNA Pol II) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitor cells (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNA Pol II initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops or promoter-promoter loops or when unlooped. Genes transitioning from repression to RNA Pol II initiation exhibit a slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNA Pol II-mediated elongation during neural cell fate transitions.

RNA m6A modification regulates cell fate transition between pluripotent stem cells and 2-cell-like cells.

N6-methyladenosine (m6A) exerts essential roles in early embryos, especially in the maternal-to-zygotic transition stage. However, the landscape and roles of RNA m6A modification during the transition between pluripotent stem cells and 2-cell-like (2C-like) cells remain elusive. Here, we utilised ultralow-input RNA m6A immunoprecipitation to depict the dynamic picture of transcriptome-wide m6A modifications during 2C-like transitions. We found that RNA m6A modification was preferentially enriched in zygotic genome activation (ZGA) transcripts and MERVL with high expression levels in 2C-like cells. During the exit of the 2C-like state, m6A facilitated the silencing of ZGA genes and MERVL. Notably, inhibition of m6A methyltransferase METTL3 and m6A reader protein IGF2BP2 is capable of significantly delaying 2C-like state exit and expanding 2C-like cells population. Together, our study reveals the critical roles of RNA m6A modification in the transition between 2C-like and pluripotent states, facilitating the study of totipotency and cell fate decision in the future.

Regulation of stomatal development by epidermal, subepidermal and long-distance signals.

Plant leaves consist of three layers, including epidermis, mesophyll and vascular tissues. Their development is meticulously orchestrated. Stomata are the specified structures on the epidermis for uptake of carbon dioxide (CO2) while release of water vapour and oxygen (O2), and thus play essential roles in regulation of plant photosynthesis and water use efficiency. To function efficiently, stomatal formation must coordinate with the development of other epidermal cell types, such as pavement cell and trichome, and tissues of other layers, such as mesophyll and leaf vein. This review summarizes the regulation of stomatal development in three dimensions (3D). In the epidermis, specific stomatal transcription factors determine cell fate transitions and also activate a ligand-receptor- MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) signaling for ensuring proper stomatal density and patterning. This forms the core regulation network of stomatal development, which integrates various environmental cues and phytohormone signals to modulate stomatal production. Under the epidermis, mesophyll, endodermis of hypocotyl and inflorescence stem, and veins in grasses secrete mobile signals to influence stomatal formation in the epidermis. In addition, long-distance signals which may include phytohormones, RNAs, peptides and proteins originated from other plant organs modulate stomatal development, enabling plants to systematically adapt to the ever changing environment.

TGF-β modulates cell fate in human ES cell-derived foregut endoderm by inhibiting Wnt and BMP signaling

Genetic differences between pluripotent stem cell lines cause variable activity of extracellular signaling pathways, limiting reproducibility of directed differentiation protocols. Here we used human embryonic stem cells (hESCs) to interrogate how exogenous factors modulate endogenous signaling events during specification of foregut endoderm lineages. We find that transforming growth factor β1 (TGF-β1) activates a putative human OTX2/LHX1 gene regulatory network which promotes anterior fate by antagonizing endogenous Wnt signaling. In contrast to Porcupine inhibition, TGF-β1 effects cannot be reversed by exogenous Wnt ligands, suggesting that induction of SHISA proteins and intracellular accumulation of Fzd receptors render TGF-β1-treated cells refractory to Wnt signaling. Subsequently, TGF-β1-mediated inhibition of BMP and Wnt signaling suppresses liver fate and promotes pancreas fate. Furthermore, combined TGF-β1 treatment and Wnt inhibition during pancreatic specification reproducibly and robustly enhance INSULIN+ cell yield across hESC lines. This modification of widely used differentiation protocols will enhance pancreatic β cell yield for cell-based therapeutic applications.

Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress

Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.

NIR‐Responsive Multifunctional Artificial Skin for Regenerative Wound Healing

AbstractEfficient wound repair with skin appendage regeneration following severe trauma poses a challenge due to the scarcity of skin grafts and decreased drug effectiveness in protease‐rich wound microenvironments. Here, a multifunctional artificial skin (NIR‐mFAS) with photothermal‐triggered drug delivery capabilities is designed to actively and comprehensively improve the regenerative potential of full‐thickness wounds. The antibacterial chitosan/silk fibroin hydrogel matrix of artificial skin, cross‐linked by electrostatic interactions, effectively encapsulates and sustains the release of epidermal growth factor (EGF) to accelerate re‐epithelialization and neovascularization by promoting the migration and proliferation of repair cells. Subsequently, the photothermal responsive polydopamine nanoparticles (PDA‐NPs) dispersed in the matrix enable precise control over the release of BMP4 under the irradiation of 1064 nm NIR, thereby inhibiting scarring by reducing myofibroblasts during the proliferative stage. Importantly, the concurrent controlled release of CHIR99021 can modulate cell fate by inducing the conversion of myofibroblasts into dermal papilla‐like cells, leading to hair follicle and sebaceous gland regeneration. The NIR‐mFAS functions as an advanced delivery system for achieving high‐quality wound healing with appendage regeneration and offers a smart therapeutic approach that can be applied to other treatments requiring coordinated delivery of multiple pharmacological agents.

SEC: A core hub during cell fate alteration.

Pol II pause release is a rate-limiting step in gene transcription, influencing various cell fate alterations. Numerous proteins orchestrate Pol II pause release, thereby playing pivotal roles in the intricate process of cellular fate modulation. Super elongation complex (SEC), a large assembly comprising diverse protein components, has garnered attention due to its emerging significance in orchestrating physiological and pathological cellular identity changes by regulating the transcription of crucial genes. Consequently, SEC emerges as a noteworthy functional complex capable of modulating cell fate alterations. Therefore, a comprehensive review is warranted to systematically summarize the core roles of SEC in different types of cell fate alterations. This review focuses on elucidating the current understanding of the structural and functional basis of SEC. Additionally, we discuss the intricate regulatory mechanisms governing SEC in various models of cell fate alteration, encompassing both physiological and pathological contexts. Furthermore, leveraging the existing knowledge of SEC, we propose some insightful directions for future research, aiming to enhance our mechanistic and functional comprehension of SEC within the diverse landscape of cell fate alterations.

Label-free metabolic optical biomarkers track stem cell fate transition in real time.

Tracking stem cell fate transition is crucial for understanding their development and optimizing biomanufacturing. Destructive single-cell methods provide a pseudotemporal landscape of stem cell differentiation but cannot monitor stem cell fate in real time. We established a metabolic optical metric using label-free fluorescence lifetime imaging microscopy (FLIM), feature extraction and machine learning-assisted analysis, for real-time cell fate tracking. From a library of 205 metabolic optical biomarker (MOB) features, we identified 56 associated with hematopoietic stem cell (HSC) differentiation. These features collectively describe HSC fate transition and detect its bifurcate lineage choice. We further derived a MOB score measuring the "metabolic stemness" of single cells and distinguishing their division patterns. This score reveals a distinct role of asymmetric division in rescuing stem cells with compromised metabolic stemness and a unique mechanism of PI3K inhibition in promoting ex vivo HSC maintenance. MOB profiling is a powerful tool for tracking stem cell fate transition and improving their biomanufacturing from a single-cell perspective.

Dysregulation of BCL-2 family proteins in blood neoplasm: therapeutic relevance of antineoplastic agent venetoclax

During cellular stress, the master regulators of intrinsic self-death (apoptosis) are BCL-2 family proteins. The BCL-2 family proteins play a key role in apoptosis and are tightly regulated via other BCL-2 family proteins, non-BCL-2 protein suppressors, and epigenetic modifications. As the name implies, these proteins possess one or two of the four BCL-2 homology domains (BH1–BH4). According to their roles, they are classified as pro-apoptotic or pro-survival proteins. BH-3-only proteins possess a single BH3 domain and are specific/key effector proteins for intracellular death commitment, particularly in the context of cell survival and programmed cell death. This delicate interplay among the BCL-2 family members is essential for maintaining the primary hemostasis, or balance, of cell fate. The anti-apoptotic proteins, such as BCL-2 and BCL-XL, promote cell survival by inhibiting apoptosis. On the other hand, the pro-apoptotic proteins, such as BAX and BAK, drive apoptosis. It ensures that cells are able to respond appropriately to various internal and external signals, ultimately determining whether a cell survives or undergoes programmed cell death. Understanding and targeting this delicate balance is a promising avenue for developing therapeutic strategies to modulate cell fate and treat various diseases. The molecular pathogenesis of BCL-2 family proteins in blood disorders involves differential expression of these components resulting in the dysregulation of the pathway contributing to cell survival and resistance to apoptosis as observed in follicular lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia, and acute myeloid leukemia. Such dysregulation is a major impediment to standard therapies and aids in chemo resistance. Studies show some promising clinical outcomes with antineoplastic agent venetoclax either as a monotherapy or in combination with other agents. This review discusses recent studies on the regulation of BCL-2 family proteins which might provide a molecular landscape for their clinical implications in blood disorders.

A single-cell transcriptome atlas reveals the trajectory of early cell fate transition during callus induction in Arabidopsis

The acquisition of pluripotent callus from somatic cells plays an important role in plant development studies and crop genetic improvement. This developmental process incorporates a series of cell fate transitions and reprogramming. However, our understanding of cell heterogeneity and mechanisms of cell fate transition during callus induction remains quite limited. Here, we report a time-series single-cell transcriptome experiment on Arabidopsis root explants that were induced in callus induction medium for 0, 1, and 4 days, and the construction of a detailed single-cell transcriptional atlas of the callus induction process. We identify the cell types responsible for initiating the early callus: lateral root primordium-initiating (LRPI)-like cells and quiescent center (QC)-like cells. LRPI-like cells are derived from xylem pole pericycle cells and are similar to lateral root primordia. We delineate the developmental trajectory of the dedifferentiation of LRPI-like cells into QC-like cells. QC-like cells are undifferentiated pluripotent acquired cells that appear in the early stages of callus formation and play a critical role in later callus development and organ regeneration. We also identify the transcription factors that regulate QC-like cells and the gene expression signatures that are related to cell fate decisions. Overall, our cell-lineage transcriptome atlas for callus induction provides a distinct perspective on cell fate transitions during callus formation, significantly improving our understanding of callus formation.

Dissecting gene activation and chromatin remodeling dynamics in single human cells undergoing reprogramming

During cell fate transitions, cells remodel their transcriptome, chromatin, and epigenome; however, it has been difficult to determine the temporal dynamics and cause-effect relationship between these changes at the single-cell level. Here, we employ the heterokaryon-mediated reprogramming system as a single-cell model to dissect key temporal events during early stages of pluripotency conversion using super-resolution imaging. We reveal that, following heterokaryon formation, the somatic nucleus undergoes global chromatin decompaction and removal of repressive histone modifications H3K9me3 and H3K27me3 without acquisition of active modifications H3K4me3 and H3K9ac. The pluripotency gene OCT4 (POU5F1) shows nascent and mature RNA transcription within the first 24h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG, conversely, has significant nascent RNA transcription only at 48h after cell fusion but, strikingly, exhibits genomic reopening early on. These findings suggest that the temporal relationship between chromatin compaction and gene activation during cellular reprogramming is gene context dependent.

SRRM2 splicing factor modulates cell fate in early development.

Embryo development is an orchestrated process that relies on tight regulation of gene expression to guide cell differentiation and fate decisions. The Srrm2 splicing factor has recently been implicated in developmental disorders and diseases, but its role in early mammalian development remains unexplored. Here, we show that Srrm2 dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness, characterised by the coexistence of cells expressing naive and formative pluripotency markers, together with extensive changes in gene expression, including genes regulated by serum-response transcription factor (SRF) and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells shows that the earliest effects of Srrm2 heterozygosity are specific alternative splicing events on a small number of genes, followed by expression changes in metabolism and differentiation-related genes. Our findings unveil molecular and cellular roles of Srrm2 in stemness and lineage commitment, shedding light on the roles of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis.

Defining a TFAP2C-centered transcription factor network during murine peri-implantation

Transcription factors (TFs) play important roles in early embryonic development, but factors regulating TF action, relationships in signaling cascade, genome-wide localizations, and impacts on cell fate transitions during this process have not been clearly elucidated. In this study, we used uliCUT&RUN-seq to delineate a TFAP2C-centered regulatory network, showing that it involves promoter-enhancer interactions and regulates TEAD4 and KLF5 function to mediate cell polarization. Notably, we found that maternal retinoic acid metabolism regulates TFAP2C expression and function by inducing the active demethylation of SINEs, indicating that the RARG-TFAP2C-TEAD4/KLF5 axis connects the maternal-to-zygotic transition to polarization. Moreover, we found that both genomic imprinting and SNP-transferred genetic information can influence TF positioning to regulate parental gene expressions in a sophisticated manner. In summary, we propose a ternary model of TF regulation in murine embryonic development with TFAP2C as the core element and metabolic, epigenetic, and genetic information as nodes connecting the pathways.

ESR2–HDA6 complex negatively regulates auxin biosynthesis to delay callus initiation in Arabidopsis leaf explants during tissue culture

Plants exhibit an astonishing ability to regulate organ regeneration upon wounding. Excision of leaf explants promotes the biosynthesis of indole-3-acetic acid (IAA), which is polar-transported to excised regions, where cell fate transition leads to root founder cell specification to induce de novo root regeneration. The regeneration capacity of plants has been utilized to develop in vitro tissue culture technologies. Here, we report that IAA accumulation near the wounded site of leaf explants is essential for callus formation on 2,4-dichlorophenoxyacetic acid (2,4-D)-rich callus-inducing medium (CIM). Notably, a high concentration of 2,4-D does not compensate for the action of IAA because of its limited efflux; rather, it lowers IAA biosynthesis via a negative feedback mechanism at an early stage of in vitro tissue culture, delaying callus initiation. The auxin negative feedback loop in CIM-cultured leaf explants is mediated by an auxin-inducible APETALA2 transcription factor, ENHANCER OF SHOOT REGENERATION 2 (ESR2), along with its interacting partner HISTONE DEACETYLASE 6 (HDA6). The ESR2–HDA6 complex binds directly to, and removes the H3ac mark from, the YUCCA1 (YUC1), YUC7, and YUC9 loci, consequently repressing auxin biosynthesis and inhibiting cell fate transition on 2,4-D-rich CIM. These findings indicate that negative feedback regulation of auxin biosynthesis by ESR2 and HDA6 interferes with proper cell fate transition and callus initiation.

Precise prediction of phase-separation key residues by machine learning

Understanding intracellular phase separation is crucial for deciphering transcriptional control, cell fate transitions, and disease mechanisms. However, the key residues, which impact phase separation the most for protein phase separation function have remained elusive. We develop PSPHunter, which can precisely predict these key residues based on machine learning scheme. In vivo and in vitro validations demonstrate that truncating just 6 key residues in GATA3 disrupts phase separation, enhancing tumor cell migration and inhibiting growth. Glycine and its motifs are enriched in spacer and key residues, as revealed by our comprehensive analysis. PSPHunter identifies nearly 80% of disease-associated phase-separating proteins, with frequent mutated pathological residues like glycine and proline often residing in these key residues. PSPHunter thus emerges as a crucial tool to uncover key residues, facilitating insights into phase separation mechanisms governing transcriptional control, cell fate transitions, and disease development.

Noggin promotes osteogenesis in human adipose-derived mesenchymal stem cells via FGFR2/Src/Akt and ERK signaling pathway

The balance between Noggin and bone morphogenetic proteins (BMPs) is important during early development and skeletal regenerative therapies. Noggin binds BMPs in the extracellular space, thereby preventing BMP signaling. However, Noggin may affect cell response not necessarily through the modulation of BMP signaling, raising the possibility of direct Noggin signaling through yet unspecified receptors. Here we show that in osteogenic cultures of adipose-derived stem cells (ASCs), Noggin activates fibroblast growth factor receptors (FGFRs), Src/Akt and ERK kinases, and it stabilizes TAZ proteins in the presence of dexamethasone. Overall, this leads ASCs to increased expression of osteogenic markers and robust mineral deposition. Our results also indicate that Noggin can induce osteogenic genes expression in normal human bone marrow stem cells and alkaline phosphatase activity in normal human dental pulp stem cells. Besides, Noggin can specifically activate FGFR2 in osteosarcoma cells. We believe our findings open new research avenues to further explore the involvement of Noggin in cell fate modulation by FGFR2/Src/Akt/ERK signaling and potential applications of Noggin in bone regenerative therapies.

Hydroxychloroquine interaction with phosphoinositide 3-kinase modulates prostate cancer growth in bone microenvironment: In vitro and molecular dynamics based approach

Patients with advanced prostate cancer (PCa) are more likely to develop bone metastases. Tumor cells thrive in the bone microenvironment, interacting with osteoblasts and osteoclasts. Given the PI3K/AKT pathway's metastatic potential and signal integration's ability to modulate cell fates in PCa development, drugs targeting this system have great therapeutic promise. Hydroxychloroquine (HCQ) is an anti-malarial medication commonly used to treat clinical conditions such as rheumatology and infectious disorders. We explored the anti-neoplastic effect of HCQ on PC3 and C4-2B cell lines in the bone microenvironment. Interestingly, HCQ treatment substantially decreases the viability, proliferation, and migration potential of PCa cells in the bone microenvironment. HCQ induces apoptosis and cell cycle arrest, even in the presence of osteoblast-secreted factors. Mechanistically, HCQ inhibited the activity of the PI3K/AKT signaling pathway, which ultimately regulates the proliferation and migration of PCa cells in the bone. The binding energy for docking HCQ with PI3K was −6.7 kcal/mol, and the complex was stabilized by hydrogen bonds, hydrophobic forces, and van der Waals forces. Molecular simulations further validated the structural integrity of the HCQ-PI3K complex without altering PI3K's secondary structure. Our findings underscore the efficacy of HCQ as a potential therapeutic agent in treating PCa.

New insights into Gasdermin D pore formation.

Gasdermin D (GSDMD) is a pore-forming protein that perforates the plasma membrane (PM) during pyroptosis, a pro-inflammatory form of cell death, to induce the unconventional secretion of inflammatory cytokines and, ultimately, cell lysis. GSDMD is activated by protease-mediated cleavage of its active N-terminal domain from the autoinhibitory C-terminal domain. Inflammatory caspase-1, -4/5 are the main activators of GSDMD via either the canonical or non-canonical pathways of inflammasome activation, but under certain stimuli, caspase-8 and other proteases can also activate GSDMD. Activated GSDMD can oligomerize and assemble into various nanostructures of different sizes and shapes that perforate cellular membranes, suggesting plasticity in pore formation. Although the exact mechanism of pore formation has not yet been deciphered, cysteine residues are emerging as crucial modulators of the oligomerization process. GSDMD pores and thus the outcome of pyroptosis can be modulated by various regulatory mechanisms. These include availability of activated GSDMD at the PM, control of the number of GSDMD pores by PM repair mechanisms, modulation of the lipid environment and post-translational modifications. Here, we review the latest findings on the mechanisms that induce GSDMD to form membrane pores and how they can be tightly regulated for cell content release and cell fate modulation.

Comparative Study of Basement-membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation.

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

Txnip regulates the Oct4-mediated pluripotency circuitry via metabolic changes upon differentiation

Thioredoxin interacting protein (Txnip) is a stress-responsive factor regulating Trx1 for redox balance and involved in diverse cellular processes including proliferation, differentiation, apoptosis, inflammation, and metabolism. However, the biological role of Txnip function in stem cell pluripotency has yet to be investigated. Here, we reveal the novel functions of mouse Txnip in cellular reprogramming and differentiation onset by involving in glucose-mediated histone acetylation and the regulation of Oct4, which is a fundamental component of the molecular circuitry underlying pluripotency. During reprogramming or PSC differentiation process, cellular metabolic and chromatin remodeling occur in order to change its cellular fate. Txnip knockout promotes induced pluripotency but hinders initial differentiation by activating pluripotency factors and promoting glycolysis. This alteration affects the intracellular levels of acetyl-coA, a final product of enhanced glycolysis, resulting in sustained histone acetylation on active PSC gene regions. Moreover, Txnip directly interacts with Oct4, thereby repressing its activity and consequently deregulating Oct4 target gene transcriptions. Our work suggests that control of Txnip expression is crucial for cell fate transitions by modulating the entry and exit of pluripotency.