Direct Cell Fate Research Articles

CRISPR screens unveil nutrient-dependent lysosomal and mitochondrial nodes impacting intestinal tissue-resident memory CD8+ T cell formation

Nutrient availability and organelle biology direct tissue homeostasis and cell fate, but how these processes orchestrate tissue immunity remains poorly defined. Here, using invivo CRISPR-Cas9 screens, we uncovered organelle signaling and metabolic processes shaping CD8+ tissue-resident memory T (TRM) cell development. TRM cells depended on mitochondrial translation and respiration. Conversely, three nutrient-dependent lysosomal signaling nodes-Flcn, Ragulator, and Rag GTPases-inhibited intestinal TRM cell formation. Depleting these molecules or amino acids activated the transcription factor Tfeb, thereby linking nutrient stress to TRM programming. Further, Flcn deficiency promoted protective TRM cell responses in the small intestine. Mechanistically, the Flcn-Tfeb axis restrained retinoic acid-induced CCR9 expression for migration and transforming growth factor β (TGF-β)-mediated programming for lineage differentiation. Genetic interaction screening revealed that the mitochondrial protein Mrpl52 enabled early TRM cell formation, while Acss1 controlled TRM cell development under Flcn deficiency-associated lysosomal dysregulation. Thus, the interplay between nutrients, organelle signaling, and metabolic adaptation dictates tissue immunity.

SMAD3 mediates the specification of human induced pluripotent stem cell-derived epicardium into progenitors for the cardiac pericyte lineage

Understanding the molecular mechanisms of epicardial epithelial-to-mesenchymal transition (EMT), particularly in directing cell fate toward epicardial derivatives, is crucial for regenerative medicine using human induced pluripotent stem cell (iPSC)-derived epicardium. Although transforming growth factor β (TGF-β) plays a pivotal role in epicardial biology, orchestrating EMT during embryonic development via downstream signaling through SMAD proteins, the function of SMAD proteins in the epicardium in maintaining vascular homeostasis or mediating the differentiation of various epicardial-derived cells (EPDCs) is not yet well understood. Our study reveals that TGF-β-independent SMAD3 expression autonomously predicts epicardial cell specification and lineage maintenance, acting as a key mediator in promoting the angiogenic-oriented specification of the epicardium into cardiac pericyte progenitors. This finding uncovers a novel role for SMAD3 in the human epicardium, particularly in generating cardiac pericyte progenitors that enhance cardiac microvasculature angiogenesis. This insight opens new avenues for leveraging epicardial biology in developing more effective cardiac regeneration strategies.

Pharmacological restriction of genomic binding sites redirects PU.1 pioneer transcription factor activity

Transcription factor (TF) DNA-binding dynamics govern cell fate and identity. However, our ability to pharmacologically control TF localization is limited. Here we leverage chemically driven binding site restriction leading to robust and DNA-sequence-specific redistribution of PU.1, a pioneer TF pertinent to many hematopoietic malignancies. Through an innovative technique, ‘CLICK-on-CUT&Tag’, we characterize the hierarchy of de novo PU.1 motifs, predicting occupancy in the PU.1 cistrome under binding site restriction. Temporal and single-molecule studies of binding site restriction uncover the pioneering dynamics of native PU.1 and identify the paradoxical activation of an alternate target gene set driven by PU.1 localization to second-tier binding sites. These transcriptional changes were corroborated by genetic blockade and site-specific reporter assays. Binding site restriction and subsequent PU.1 network rewiring causes primary human leukemia cells to differentiate. In summary, pharmacologically induced TF redistribution can be harnessed to govern TF localization, actuate alternate gene networks and direct cell fate.

Stochastic Modeling of Biophysical Responses to Perturbation.

Recent advances in high-throughput, multi-condition experiments allow for genome-wide investigation of how perturbations affect transcription and translation in the cell across multiple biological entities or modalities, from chromatin and mRNA information to protein production and spatial morphology. This presents an unprecedented opportunity to unravel how the processes of DNA and RNA regulation direct cell fate determination and disease response. Most methods designed for analyzing large-scale perturbation data focus on the observational outcomes, e.g., expression; however, many potential transcriptional mechanisms, such as transcriptional bursting or splicing dynamics, can underlie these complex and noisy observations. In this analysis, we demonstrate how a stochastic biophysical modeling approach to interpreting high-throughout perturbation data enables deeper investigation of the 'how' behind such molecular measurements. Our approach takes advantage of modalities already present in data produced with current technologies, such as nascent and mature mRNA measurements, to illuminate transcriptional dynamics induced by perturbation, predict kinetic behaviors in new perturbation settings, and uncover novel populations of cells with distinct kinetic responses to perturbation.

Inhibition of Atm and Atr kinases affects embryo development and the DNA damage response in sterlet (Acipenser ruthenus)

Embryo development is regulated by numerous interconnected pathways that direct cell fate, differentiation, and genetic stability. During development, these pathways can be altered by exogenous factors, such as pollutants, or endogenous factors, such as replication stress. Ataxia telangiectasia mutated (Atm) and ataxia telangiectasia and Rad3-related (Atr) kinases play critical roles in regulating the cell cycle, the DNA damage response (DDR), DNA repair, checkpoint activation, and apoptosis. In cells with damaged DNA, these kinases can slow the cell cycle to provide the necessary time and ability for DNA repair. In this study, we investigated the roles of Atm and Atr in embryo development and DDR in the sterlet (Acipenser ruthenus). Sterlets belong to the Acipenseridae family, one of the most threatened groups of species. Thus, understanding their genetics, biology, embryogenesis, and conservation is crucial. Furthermore, the sterlet is a significant aquaculture species that represents an intriguing model for studying polyploidy and genome plasticity. In our research, we examined the effects of chemical inhibition of Atm and Atr on sterlet embryo development in the absence and presence of the genotoxicant camptothecin (CPT). Our findings indicated that in the absence of genotoxic challenge, Atr autophosphorylation increases between 1 and 3 days post-fertilization (dpf) and decreases by 5 dpf, illustrating the involvement of Atr in the response to replication stress in rapidly differentiating tissues during the early stages of embryo development. Conversely, Atm inhibition was associated with a dose-dependent reduction in embryo viability, highlighting its importance in normal embryo development. When sterlet embryos were exposed to CPT, both Atm and Atr were involved in DDR and the activation of apoptosis. Notably, when both kinases were inhibited simultaneously, the embryos lost their ability to induce apoptosis and mitigate DNA damage, resulting in 100% embryonic mortality. These results suggest that Atm and Atr have distinct functions during normal embryo development, but they can partially complement each other in DDR.

Enhancing the Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes with an n-Type Organic Semiconductor Coating.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are a promising cell source for cardiac regenerative medicine and in vitro modeling. However, hPSC-CMs exhibit immature structural and functional properties compared with adult cardiomyocytes. Various electrical, mechanical, and biochemical cues have been applied to enhance hPSC-CM maturation but with limited success. In this work, we investigated the potential application of the semiconducting polymer poly{[N,N'-bis(2-octyldodecyl)-naphthalene-1,4,5,8-bis(dicarboximide)-2,6-diyl]-alt-5,5'-(2,2'-bithiophene)} (P(NDI2OD-T2)) as a light-sensitive material to stimulate hPSC-CMs optically. Our results indicated that P(NDI2OD-T2)-mediated photostimulation caused cell damage at irradiances applied long-term above 36 μW/mm2 and did not regulate cardiac monolayer beating (after maturation) at higher intensities applied in a transient fashion. However, we discovered that the cells grown on P(NDI2OD-T2)-coated substrates showed significantly enhanced expression of cardiomyocyte maturation markers in the absence of a light exposure stimulus. A combination of techniques, such as atomic force microscopy, scanning electron microscopy, and quartz crystal microbalance with dissipation monitoring, which we applied to investigate the interface of the cell with the n-type coating, revealed that P(NDI2OD-T2) impacted the nanostructure, adsorption, and viscoelasticity of the Matrigel coating used as a cell adhesion promoter matrix. This modified cellular microenvironment promoted the expression of cardiomyocyte maturation markers related to contraction, calcium handling, metabolism, and conduction. Overall, our findings demonstrate that conjugated polymers such as P(NDI2OD-T2) can be used as passive coatings to direct stem cell fate through interfacial engineering of cell growth substrates.

Inorganic ions activate lineage-specific gene regulatory networks

Inorganic biomaterials have been shown to direct cellular responses, including cell−cell and cell−matrix interactions. Notably, ions released from these inorganic biomaterials play a vital role in defining cell identity, and promoting tissue-specific functions. However, the effect of inorganic ions on cellular functions have yet to be investigated at the transcriptomic level, representing a critical knowledge gap in the development of next-generation bioactive materials. To address this gap, we investigated the impact of various inorganic ions including silver, copper, titanium, and platinum on human mesenchymal stem cells (hMSCs). Our finding showed that silver and copper induce osteogenic and chondrogenic differentiation respectively, through enrichment of lineage-specific gene expression program. In particular, silver effectively induced Wingless/Integrated (Wnt) and mitogen-activated protein kinase (MAPK) signaling, which are vital for osteogenesis. On the other hand, copper specifically stimulated Transforming growth factor beta (TGFβ) signaling, while suppressing Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling, thereby promoting chondrogenesis. In contrast, platinum, and tantalum, ions didn't stimulate regenerative responses. Together, our findings highlight the potential of inorganic biomaterials in tissue regeneration strategies, which currently rely largely on growth factors and small molecule therapeutics. Statement of significanceThis research emphasizes the critical role of bioactive inorganic ions in controlling lineage-specific gene expression patterns in mesenchymal stem cells, effectively modulating the transcriptome landscape and directing cell fate. The study lays the foundation for a systematic database of biomaterial candidates and their effects on cellular functions, which will ultimately streamline the translation of new biomaterials into clinical applications.

Guiding Stem Cell Tenogenesis by Modulation of Growth Factor Signaling and Cell‐Scale Biophysical Cues in Bioengineered Constructs

AbstractTendon injuries and tendinopathies are increasingly prevalent health problems currently lacking effective treatments. Tissue engineering offers promising strategies to boost the low innate regenerative ability of tendons. Within this context, the simultaneous leveraging of both physical and biochemical cues by engineered scaffolding systems can be explored to promote a stronger tenogenic response from stem cells. Here, molecularly imprinted polymeric nanoparticles (MINPs) against transforming growth factor (TGF)‐β3 are combined with bioinspired anisotropic hydrogels to produce tenogenesis‐inductive constructs. MINPs are first solid phase‐imprinted against a TGF‐β3 epitope, achieving an affinity comparable to monoclonal antibodies. MINPs and magnetically‐responsive microfibers are then encapsulated together with adipose‐derived stem cells within gelatin‐based hydrogels, applying a magnetostatic field during gelation to align the microfibers. The created anisotropic microstructure guides cell growth and elongation unidirectionally, while MINPs act as artificial receptors for TGF‐β3, potentiating its paracrine action in the cellular microenvironment. The combination of both stimuli proves effective at increasing TGF‐β signaling, which promotes the expression of tendon‐associated genes and corresponding protein synthesis, suggesting that microstructural cues and biomolecule sequestration act in tandem to direct cell fate commitment. Overall, this system recapitulates several elements of tendon development, constituting a promising strategy for the regeneration of this tissue.

Stem cell mTOR signaling directs region-specific cell fate decisions during intestinal nutrient adaptation.

The adult intestine is a regionalized organ, whose size and cellular composition are adjusted in response to nutrient status. This involves dynamic regulation of intestinal stem cell (ISC) proliferation and differentiation. How nutrient signaling controls cell fate decisions to drive regional changes in cell-type composition remains unclear. Here, we show that intestinal nutrient adaptation involves region-specific control of cell size, cell number, and differentiation. We uncovered that activation of mTOR complex 1 (mTORC1) increases ISC size in a region-specific manner. mTORC1 activity promotes Delta expression to direct cell fate toward the absorptive enteroblast lineage while inhibiting secretory enteroendocrine cell differentiation. In aged flies, the ISC mTORC1 signaling is deregulated, being constitutively high and unresponsive to diet, which can be mitigated through lifelong intermittent fasting. In conclusion, mTORC1 signaling contributes to the ISC fate decision, enabling regional control of intestinal cell differentiation in response to nutrition.

A Metabolic-Epigenetic Mechanism Directs Cell Fate and Therapeutic Sensitivity in Breast Cancer.

Over the past decade, studies have increasingly shed light on a reciprocal relationship between cellular metabolism and cell fate, meaning that a cell's lineage both drives and is governed by its specific metabolic features. A recent study by Zhang and colleagues, published in Cell Metabolism, describes a novel metabolic-epigenetic regulatory axis that governs lineage identity in triple-negative breast cancer (TNBC). Among the key findings, the authors demonstrate that the metabolic enzyme pyruvate kinase M2 (PKM2) directly binds to the histone methyltransferase enhancer of zeste homolog 2 (EZH2) in the nucleus to silence expression of a set of genes that includes the mitochondrial carnitine transporter SLC16A9. Perturbation of this metabolic-epigenetic regulatory mechanism induces a metabolic shift away from glycolysis and toward fatty acid oxidation. The ensuing influx of carnitine facilitates the deposition of the activating epigenetic mark H3K27Ac onto the promoter of GATA3, driving a committed luminal lineage state. Importantly, this metabolic-epigenetic axis represents a potentially targetable vulnerability for the treatment of TNBC, a subtype that currently lacks effective therapeutic strategies. These findings lend further support for the paradigm shift underlying our understanding of cancer metabolism: that a cellular fuel source functions not only to provide energy but also to direct the epigenetic regulation of cell fate.

Opposite Mechanical Preference of Bone/Nerve Regeneration in 3D-Printed Bioelastomeric Scaffolds/Conduits Consistently Correlated with YAP-Mediated Stem Cell Osteo/Neuro-Genesis.

To systematically unveil how substrate stiffness, a critical factor in directing cell fate through mechanotransduction, correlates with tissue regeneration, novel biodegradable and photo-curable poly(trimethylene carbonate) fumarates (PTMCFs) for fabricating elastomeric 2D substrates and 3D bone scaffolds/nerve conduits, are presented. These substrates and structures with adjustable stiffness serve as a unique platform to evaluate how this mechanical cue affects the fate of human umbilical cord mesenchymal stem cells (hMSCs) and hard/soft tissue regeneration in rat femur bone defect and sciatic nerve transection models; whilst, decoupling from topographical and chemical cues. In addition to a positive relationship between substrate stiffness (tensile modulus: 90-990kPa) and hMSC adhesion, spreading, and proliferation mediated through Yes-associated protein (YAP), opposite mechanical preference is revealed in the osteogenesis and neurogenesis of hMSCs as they are significantly enhanced on the stiff and compliant substrates, respectively. In vivo tissue regeneration demonstrates the same trend: bone regeneration prefers the stiffer scaffolds; while, nerve regeneration prefers the more compliant conduits. Whole-transcriptome analysis further shows that upregulation of Rho GTPase activity and the downstream genes in the compliant group promote nerve repair, providing critical insight into the design strategies of biomaterials for stem cell regulation and hard/soft tissue regeneration through mechanotransduction.

Composite Spheroid-Laden Bilayer Hydrogel for Engineering Three-Dimensional Osteochondral Tissue.

A combination of hydrogels and stem cell spheroids has been used to engineer three-dimensional (3D) osteochondral tissue, but precise zonal control directing cell fate within the hydrogel remains a challenge. In this study, we developed a composite spheroid-laden bilayer hydrogel to imitate osteochondral tissue by spatially controlled differentiation of human adipose-derived stem cells. Meticulous optimization of the spheroid-size and mechanical strength of gelatin methacryloyl (GelMA) hydrogel enables the cells to homogeneously sprout within the hydrogel. Moreover, fibers immobilizing transforming growth factor beta-1 (TGF-β1) or bone morphogenetic protein-2 (BMP-2) were incorporated within the spheroids, which induced chondrogenic or osteogenic differentiation of cells in general media, respectively. The spheroids-filled GelMA solution was crosslinked to create the bilayer hydrogel, which demonstrated a strong interfacial adhesion between the two layers. The cell sprouting enhanced the adhesion of each hydrogel, demonstrated by increase in tensile strength from 4.8 ± 0.4 to 6.9 ± 1.2 MPa after 14 days of culture. Importantly, the spatially confined delivery of BMP-2 within the spheroids increased mineral deposition and more than threefold enhanced osteogenic genes of cells in the bone layer while the cells induced by TGF-β1 signals were apparently differentiated into chondrocytes within the cartilage layer. The results suggest that our composite spheroid-laden hydrogel could be used for the biofabrication of osteochondral tissue, which can be applied to engineer other complex tissues by delivery of appropriate biomolecules.

3D BIOMIMETIC CONSTRUCTS STEER STEM CELL COMMITMENT BY SYNERGISTIC MODULATION OF BIOPHYSICAL CUES AND GROWTH FACTOR SIGNALLING

Tendon diseases are prevalent health concerns for which current therapies present limited success, in part due to the intrinsically low regenerative ability of tendons. Therefore, tissue engineering presents a potential to improve this outcome. Here, we hypothesize that a concurrent control over both biophysical and biochemical stimuli will boost the tenogenic commitment of stem cells, thus promoting regeneration. To achieve this, we combine molecularly imprinted nanoparticles (MINPs), which act as artificial amplifiers for endogenous growth factor (GF) activity, with bioinspired anisotropic hydrogels2 to manufacture 3D tenogenic constructs. MINPs were solid phase-imprinted using a TGF-β3 epitope as template and their affinity for the target was assessed by SPR and dot blot. Magnetically-responsive microfibers were produced by cryosectioning electrospun meshes containing iron oxide nanoparticles. The constructs were prepared by encapsulating adipose tissue-derived stem cells (ASCs), microfibers, and MINPs within gelatin hydrogels, while aligning the microfibers with an external magnetostatic field during gelation. This allows an effective modulation of hydrogel fibrillar topography, mimicking the native tissue's anisotropic architecture. Cell responses were analyzed by multiplex immunoassay, quantitative polymerase chain reaction, and immunocytochemistry. MINPs showed an affinity for the template comparable to monoclonal antibodies. Encapsulated ASCs acquired an elongated shape and predominant orientation along the alignment direction. Cellular studies revealed that combining MINPs with aligned microfibers increased TGF-β signaling via non-canonical Akt/ERK pathways and upregulated tendon-associated gene expression, contrasting with randomly oriented gels. Immunostaining of tendon-related proteins presented analogous outcomes, corroborating our hypothesis.Our results thus demonstrate that microstructural cues and biological signals synergistically direct stem cell fate commitment, suggesting that this strategy holds potential for improving tendon healing and might be adaptable for other biological tissues. The proposed concept highlights the GF-sequestering ability of MINPs which allows a cost-effective alternative to recombinant GF supplementation, potentially decreasing the translational costs of tissue engineering strategies.Acknowledgements: The authors acknowledge the funding from the European Union's Horizon 2020 under grant No. 772817; from FCT/MCTES for scholarships PD/BD/143039/2018 & COVID/BD/153025/2022 (S.P.B.T.), and PD/BD/129403/2017 (S.M.B.), co-financed by POCH and NORTE 2020, under the Portugal 2020 partnership agreement through the European Social Fund, for contract 2020.03410.CEECIND (R.M.A.D.) and project 2022.05526.PTDC; and from Xunta de Galicia for grant ED481B2019/025 (A.P.).

Mind the Viscous Modulus: The Mechanotransductive Response to the Viscous Nature of Isoelastic Matrices Regulates Stem Cell Chondrogenesis.

The design of hydrogels as mimetics of tissues' matrices typically disregards the viscous nature of native tissues and focuses only on their elastic properties. In the case of stem cell chondrogenesis, this has led to contradictory results, likely due to unreported changes in the matrices' viscous modulus. Here, by employing isoelastic matrices with Young's modulus of ≈12kPa, variations in viscous properties alone (i.e., loss tangent between 0.1 and 0.25) are demonstrated to be sufficient to drive efficient growth factor-free chondrogenesis of human mesenchymal stem cells, both in 2D and 3D cultures. The increase of the viscous component of RGD-functionalized polyacrylamide or polyethylene glycol maleimide hydrogels promotes a phenotype with reduced adhesion, alters mechanosensitive signaling, and boosts cell-cell contacts. In turn, this upregulates the chondrogenic transcription factor SOX9 and supports neocartilage formation, demonstrating that the mechanotransductive response to the viscous nature of the matrix can be harnessed to direct cell fate.

EPCO-41. MICRORNA-MEDIATED DIRECT NEURONAL REPROGRAMMING OF GLIOBLASTOMAS

Abstract Neuron-specific microRNAs miR-9/9* and miR-124 (miR-9/9*-124) are potent neurogenic molecules whose ectopic expression in non-neuronal somatic cells effectively induces direct cell fate conversion into a neuronal fate. Our recent work indicates that the fate transition occurs in distinct steps where miR-9/9*-124 first represses core gene regulatory networks to erase the original non-neuronal cell identity and subsequently activates the neuronal program to confer neuronal fate. Extending these findings, we reason that repression of similar gene regulatory networks by miR-9/9*-124 can also induce fate erasure in cancerous cells using glioblastoma as a paradigm. Glioblastoma multiforme (GBM) is a type of aggressive brain tumor with poor prognosis. Instead of having specific mutation profiles, GBMs present a global genetic network dysregulation which gives rise to a neural stem cell or progenitor cell-like state resembling embryonic neural development. Whereas miR-9/9*-124 plays a key role in inducing mitotic exit and orchestrating neurogenesis during development and neuronal reprogramming, we hypothesize that miR-9/9*-124 will instruct neuronal reprogramming of GBMs. Our results so far demonstrate that miR-9/9*-124 indeed exerts reprogramming activities in patient-derived GBM cell lines. Ectopic expression of miR-9/9*-124 in vitro readily induces GBM cells to exit cell cycle and promotes neuronal identity over time. In GBM intracerebral xenograft mouse model, mice receiving GBM cells overexpressing miR-9/9*-124 were able to survive longer than those with GBMs overexpressing non-specific microRNAs. We are currently carrying out transcriptomic and chromatin accessibility analyses to infer the epigenetic switch as GBM cells are reprogrammed to neurons. Based on these studies, we will further examine whether the GBM cells undergo irreversible fate erasure and are reprogrammed into a stable post-mitotic neuronal state. Our studies will provide insights into the plasticity of GBM fate that can be controlled by microRNAs and the key downstream regulatory nodes underlying fate transition of GBM to permanent post-mitotic state.

Targeting ACC1 in T cells ameliorates psoriatic skin inflammation

Psoriasis is a chronic inflammatory skin disease driven by the IL-23/IL-17 axis. It results from excessive activation of effector T cells, including T helper (Th) and cytotoxic T (Tc) cells, and is associated with dysfunctional regulatory T cells (Tregs). Acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme of fatty acid synthesis (FAS), directs cell fate decisions between Th17 and Tregs and thus could be a promising therapeutic target for psoriasis treatment. Here, we demonstrate that targeting ACC1 in T cells by genetic ablation ameliorates skin inflammation in an experimental model of psoriasis by limiting Th17, Tc17, Th1, and Tc1 cells in skin lesions and increasing the frequency of effector Tregs in skin-draining lymph nodes (LNs).Key messagesACC1 deficiency in T cells ameliorates psoriatic skin inflammation in mice.ACC1 deficiency in T cells reduces IL-17A-producing Th17/Tc17/dysfunctional Treg populations in psoriatic lesions.ACC1 deficiency in T cells restrains IFN-γ-producing Th1/Tc1 populations in psoriatic skin lesions and skin-draining LNs.ACC1 deficiency promotes activated CD44+CD25+ Tregs and effector CD62L-CD44+ Tregs under homeostasis and psoriatic conditions.

Mitochondrial integrated stress response controls lung epithelial cell fate.

Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1-6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.

Lysosomal cystine export regulates mTORC1 signaling to guide kidney epithelial cell fate specialization

Differentiation is critical for cell fate decisions, but the signals involved remain unclear. The kidney proximal tubule (PT) cells reabsorb disulphide-rich proteins through endocytosis, generating cystine via lysosomal proteolysis. Here we report that defective cystine mobilization from lysosomes through cystinosin (CTNS), which is mutated in cystinosis, diverts PT cells towards growth and proliferation, disrupting their functions. Mechanistically, cystine storage stimulates Ragulator-Rag GTPase-dependent recruitment of mechanistic target of rapamycin complex 1 (mTORC1) and its constitutive activation. Re-introduction of CTNS restores nutrient-dependent regulation of mTORC1 in knockout cells, whereas cell-permeant analogues of L-cystine, accumulating within lysosomes, render wild-type cells resistant to nutrient withdrawal. Therapeutic mTORC1 inhibition corrects lysosome and differentiation downstream of cystine storage, and phenotypes in preclinical models of cystinosis. Thus, cystine serves as a lysosomal signal that tailors mTORC1 and metabolism to direct epithelial cell fate decisions. These results identify mechanisms and therapeutic targets for dysregulated homeostasis in cystinosis.

Mechanobiology-informed biomaterial and tissue engineering strategies for influencing skeletal stem and progenitor cell fate.

Skeletal stem and progenitor cells (SSPCs) are the multi-potent, self-renewing cell lineages that form the hematopoietic environment and adventitial structures of the skeletal tissues. Skeletal tissues are responsible for a diverse range of physiological functions because of the extensive differentiation potential of SSPCs. The differentiation fates of SSPCs are shaped by the physical properties of their surrounding microenvironment and the mechanical loading forces exerted on them within the skeletal system. In this context, the present review first highlights important biomolecules involved with the mechanobiology of how SSPCs sense and transduce these physical signals. The review then shifts focus towards how the static and dynamic physical properties of microenvironments direct the biological fates of SSPCs, specifically within biomaterial and tissue engineering systems. Biomaterial constructs possess designable, quantifiable physical properties that enable the growth of cells in controlled physical environments both in-vitro and in-vivo. The utilization of biomaterials in tissue engineering systems provides a valuable platform for controllably directing the fates of SSPCs with physical signals as a tool for mechanobiology investigations and as a template for guiding skeletal tissue regeneration. It is paramount to study this mechanobiology and account for these mechanics-mediated behaviors to develop next-generation tissue engineering therapies that synergistically combine physical and chemical signals to direct cell fate. Ultimately, taking advantage of the evolved mechanobiology of SSPCs with customizable biomaterial constructs presents a powerful method to predictably guide bone and skeletal organ regeneration.

75-OR: Cold-Responsive Proteolytic Rewiring of Mitochondria Safeguards White Fat Thermogenic Remodeling

Mitochondrial proteases are emerging as key regulators of mitochondrial plasticity acting both as protein quality surveillance and regulatory enzymes. It remains unclear, however, whether the regulated mitochondrial proteolysis is mechanistically linked to cell identity switch such as white-to-beige conversion of adipocytes. Here we report that cold-responsive mitochondrial proteolysis is a prerequisite for conversion from white-to-beige adipocytes during white adipose tissue (WAT) browning. MitoTimer reporter and chemical thiol probe analysis reveal that thermogenic stimulation selectively promotes mitochondrial proteostasis in WAT via mitochondrial protease LONP1 both in vivo and in vitro. Disruption of LONP1-dependent proteolysis substantially impairs cold or b3-AR agonist induced adipocyte identity programming, evidenced by the remarkably attenuated formation of UCP1+ multilocular adipocytes in IWAT from Lonp1 f/f/Adipoq-Cre (LONP1 AKO) mice. LONP1 AKO mice exposed to cold had lower body temperature, elevated serum TG and FFA levels. Chow-fed LONP1 AKO mice also developed glucose intolerance. “AdipoChaser” system “pulse-chase” lineage tracing experiments confirm that LONP1 ablation markedly blocks the direct cell fate programming of pre-existing mature adipocytes. Importantly, augmented LONP1 expression corrects aging-related impairments in white-to-beige adipocytes conversion. 10-month-old adipocyte-specific Lonp1 overexpression mice had significantly lower body weight and fat mass as a result of elevated energy expenditure. Thus, the identification of LONP1 as a critical safeguard of WAT beiging holds promise for translational applications in enhancing white-to-beige adipocytes conversion capacity relevant to a variety of metabolic disease states and during aging. Disclosure T.Fu: None. W.Sun: None. Z.Zhou: None. Z.Xu: None. M.Yan: None. L.Yang: None. Y.Yin: None. Z.Gan: None.