Myelodysplastic neoplasms (MDS) disrupt bone marrow hematopoiesis, yet clinical assessment relies largely on blast enumeration and qualitative morphology, which incompletely capture marrow architecture and disease state. We applied whole-slide multiplex immunofluorescence imaging with single-cell phenotyping to map bone marrow microarchitecture in MDS. Diagnostic biopsies (n = 36), longitudinal treatment samples (n = 29), precursor states (n = 13), and normal controls (n = 21) were analyzed, comprising >5 million spatially resolved cells. MDS marrow exhibited coordinated, genotype-imprinted architectural remodeling, including altered progenitor composition and spatial patterning, disrupted erythroid island organization, and displacement of hematopoietic stem and progenitor cells from perivascular niches. Interrogation of 82 cellular and spatial features yielded a composite Microarchitectural Perturbation Score (MDS-MAPS), derived from diagnostic samples and fixed prior to longitudinal analyses. In leave-one-patient-out cross-validation, MDS-MAPS discriminated remission from active disease more accurately than blast percentage (AUC 0.883 vs 0.660) and distinguished low-blast MDS from clonal cytopenia of undetermined significance (CCUS) (AUC 0.815). Mixed-effects modeling showed MAPS decreased in remission statistically independent of blast burden, with architectural normalization during remission and re-emergence at relapse. These findings define quantitative bone marrow architecture as a dynamic tissue-state biomarker that complements molecular and blast-based assessment in MDS.

Atomic force microscopy (AFM) provides quantitative capabilities for mapping the mechanical properties of living cells with nanometer resolution, yet its potential for monitoring a range of cell mechanical states remains largely underexplored. Here, we employed AFM to map the mechanical state progression of endothelial cells (ECs) across physiological and stress conditions. Distinct mechanical signatures emerged for each physiological state: cells stiffen during division, soften in response to cell stress such as inflammation, and exhibit mechanical collapse when cytoskeletal integrity is compromised. High-resolution AFM imaging of the cytoskeletal architecture revealed changes in its organization and coherency. By simultaneously tracking the mechanical and topographical changes, AFM enables the prediction of cellular viability and functional state in real-time. AFM measurements provide a real-time assessment of endothelial cell state in vitro, with broader implications for understanding endothelial dysfunction in vascular disease and enabling new tools in tissue engineering.

Dysfunction of specialized adipose endothelial cells has been implicated in the metabolic derangements observed in individuals with obesity and type 2 diabetes. In this issue of Nature Metabolism, AlZaim et al. provide a single-cell molecular atlas of the human subcutaneous adipose tissue and highlight the possible contributions of adipose endothelial cells to adipose tissue pathology.

Gastric insulin-secreting organoids (GINS) represent a promising source of β-like cells for type 1 diabetes (T1D) therapy. In same-donor comparisons with induced pluripotent stem cell-derived islets (iPSC-islets), GINS displayed robust glucose responsiveness and reduced expression of key T1D autoantigens. Importantly, GINS exhibited decreased susceptibility to cytotoxicity mediated by engineered HLA-matched preproinsulin-specific effector T cells (Avatar Teffs) and a distinct transcriptional profile enriched for immune-modulatory and stress-adaptive gene programs. To enhance immune evasion, we engineered gastric stem cells to overexpress Programmed Death Ligand 1 (PD-L1) in an inducible manner. PD-L1 + GINS maintained normal functionality, while exhibiting improved survival under allogeneic Avatar Teff challenge in a MHC class I-independent fashion. We evaluated PD-L1-mediated protection against autologous Avatar Teff attack using an endothelialized microfluidic platform recapitulating physiologic immune interactions. T cells show reduced infiltration into PD-L1⁺ GINS, resulting in significantly higher organoid viability compared to control GINS. Together, these findings identify GINS as a functional and engineerable β-like cell platform with intrinsic hypoimmunogenic features, and support PD-L1 engineering as a strategy to enhance immune protection for both allogeneic and autologous transplantation in T1D.

Mammalian spermatogonial stem cells (SSCs) sustain male fertility through continuous self-renewal and differentiation, leading to the production of haploid spermatozoa throughout adulthood. However, SSCs are vulnerable to genotoxic drugs, and patients receiving chemotherapy face a high risk of germline instability and infertility. The molecular mechanisms and cellular pathways that choreograph SSC recovery after chemotherapeutic insult remain unknown. Previously, we identified SPRY4 as an ERK-dependent negative feedback regulator of growth factor signaling that is critical for preservation of stem cell activity in cultured mouse SSCs. Here, we demonstrate that following alkylating agent busulfan (BU)-induced injury in adult mice, germline-specific Spry4 gene deletion (Spry4G-KO) reduces stem cell regeneration with an enhanced genotoxic stress response and differentiation with rapidly enhanced nuclear ERK1/2 activity in undifferentiated (Aundiff) spermatogonia (including SSCs). Genes essential for stem cell maintenance, including Id1 and Cxcl12, were dysregulated by loss of Spry4. Furthermore, the MEK1/2 inhibitor PD0325901, but not mTORC1 inhibitor rapamycin, was sufficient to promote spermatogonial proliferation in Spry4G-KO testis 10 days post-BU treatment. Notably, the restoration of both spermatogonia pool and fertility was delayed in adult Spry4G-KO males long-term after injury. In summary, germline-specific deletion of Spry4 results in hyper-activation of the MAPK/ERK pathway in Aundiff spermatogonia, reducing spermatogonial genome integrity, unleashing excessive spermatogenesis after germline damage, and ultimately impairing germline regeneration in adult males. Our study indicates an essential role for SPRY4-ERK signaling as a molecular checkpoint in securing SSC recovery upon chemotherapy drug-induced germline damage, revealing how stem cells normally withstand environmental stress.

Liver fibrosis is a prominent pathological process contributing to death from hepatic diseases, including metabolic dysfunction-associated steatohepatitis (MASH). There is limited treatment for liver fibrosis. Here, we find that upregulation of Rho-associated coiled-coil containing kinase 2 (ROCK2) in liver endothelial cells (ECs) and perivascular hepatic stellate cells (HSCs) causes vascular niche dysfunction and triggers pro-fibrotic angiocrine signaling. Based on the vascular druggable target ROCK2, we developed its selective inhibitor showing anti-fibrotic potency in preclinical models and human patients. The ROCK2-selective inhibitor TDI01 restored vascular phenotype and alleviated fibrosis in rodent and minipig MASH models. A phase 1 clinical trial (ChiCTR2200058868) of TDI01 demonstrated its favorable pharmacokinetics and safety in humans. An extended clinical trial (ChiCTR2400082056) showed a trend toward reducing liver fibrosis in five of six patients after TDI01 treatment. Thus, we discover vascular ROCK2 as a pro-fibrotic target, and development of an inhibitor selectively targeting angiocrine ROCK2 may provide a treatment of liver fibrosis in human patients.

Decoding the gene regulatory mechanisms mediating self-renewal of hematopoietic stem cells (HSCs) during their amplification in the fetal liver (FL) is relevant for advancing therapeutic applications aiming to expand transplantable HSCs, a long-standing challenge. Here, to explore intrinsic and extrinsic regulation of self-renewal in FL-HSCs at the single cell level, we engineered a culture platform designed to recapitulate the FL endothelial niche, which supports the amplification of serially engraftable HSCs ex vivo. Leveraging this platform in combination with single cell index flow cytometry, serial transplantation assays, and single cell RNA-sequencing, we elucidated previously unrecognized heterogeneity in immunophenotypically defined FL-HSCs and demonstrated that differentiation latency and transcriptional signatures of biosynthetic dormancy are distinguishing properties of self-renewing FL-HSCs with capacity for serial, long-term multilineage hematopoietic reconstitution. Altogether, our findings provide key insights into HSC expansion and generate a novel resource for future exploration of the intrinsic and niche-derived signaling pathways that support FL-HSC self-renewal.

Microfluidic platforms offer a powerful approach for ultimately replicating vascularization in vitro, enabling precise microscale control and manipulation of physical parameters. Despite these advances, the real-time ability to monitor and quantify mechanical forces—particularly pressure—within microfluidic environments remains constrained by limitations in cost and compatibility across diverse device architectures. Our work presents an advanced experimental module for quantifying pressure within a vascularizing microfluidic platform. Equipped with an integrated Arduino microcontroller and image monitoring, the system facilitates real-time remote monitoring to access temporal pressure and flow dynamics within the device. This setup provides actionable insights into the hemodynamic parameters driving vascularization in vitro. In-line pressure sensors, interfaced through I2C communication, are employed to precisely record inlet and outlet pressures during critical stages of microvasculature tubulogenesis. Flow measurements are obtained by analyzing changes in reservoir volume over time (dV/dt), correlated with the change in pressure over time (dP/dt). This quantitative assessment of various pressure conditions in a microfluidic platform offers insights into their impact on microvasculature perfusion kinetics. Data acquisition can help inform and finetune functional vessel network formation and potentially enhance the durability, stability, and reproducibility of engineered in vitro platforms for organoid vascularization in regenerative medicine.

During development, endothelial cells (ECs) undergo an extraordinary specialization by which generic capillary microcirculatory networks spanning from arteries to veins transform into patterned organotypic zonated blood vessels. These capillary ECs become specialized to support the cellular and metabolic demands of each specific organ, including supplying tissue-specific angiocrine factors that orchestrate organ development, maintenance of organ-specific functions and regeneration of injured adult organs. Here, we illustrate the mechanisms by which microenvironmental signals emanating from non-vascular niche cells induce generic ECs to acquire specific inter-organ and intra-organ functional attributes. We describe how perivascular, parenchymal and immune cells dictate vascular heterogeneity and capillary zonation, and how this system is maintained through tissue-specific signalling activated by vasculogenic and angiogenic factors and deposition of matrix components. We also discuss how perturbation of organotypic vascular niche cues lead to erasure of EC signatures, contributing to the pathogenesis of disease processes. We also describe approaches that use reconstitution of tissue-specific signatures of ECs to promote regeneration of damaged organs. Endothelial cells (ECs) undergo organ-specific specialization, driven by microenvironmental cues, to form patterned vascular networks. This article discusses mechanisms driving vascular inter-organ and intra-organ EC heterogeneity, crosstalk between ECs and neighbouring cells, and the therapeutic potential of engineering ECs.

Tissue-specific endothelial cells (ECs) are critical for the homeostasis of pancreatic islets and most other tissues. In vitro recapitulation of islet biology and therapeutic islet transplantation both require adequate vascularization, which remains a challenge. Using human reprogrammed vascular ECs (R-VECs), human islets were functionally vascularized in vitro, demonstrating responsive, dynamic glucose-stimulated insulin secretion and Ca2+ influx. Subcutaneous transplantation of islets with R-VECs reversed hyperglycemia in diabetic mice, with high levels of human insulin detected within recipient serum and relapses of hyperglycemia following graft removal. Examination of retrieved grafts demonstrated that engrafted human islets were mainly vascularized by the cotransplanted R-VECs, which had anastomosed with the host microcirculation. Notably, single-cell RNA-sequencing revealed that R-VECs, when cocultured with islets, acquired islet EC-specific characteristics. Together, R-VECs establish an adaptable vascular niche that supports islet homeostasis both in vitro and in vivo.

The pancreatic islet, the only type of tissue that secretes insulin in response to elevated blood glucose, plays a vital role in diabetes development and treatment. While various islet vascularization strategies have been developed, they have been hindered by major limitations such as relying on pre-patterning and the inability to span long distances. Furthermore, few strategies have demonstrated robust enough vascularization in vivo to support therapeutic subcutaneous islet transplantation. Using adaptive endothelial cells (ECs) reprogrammed by transient expression of the ETS Variant Transcription Factor 2 (ETV-2) gene, we have physiologically vascularized human islets within a generic microchamber and have achieved functional engraftment of human islets in the subcutaneous space of mice. Such adaptive ECs, which we term reprogrammed vascular ECs (R-VECs), have been proven to be a suitable tool for both in vitro disease modeling and in vivo transplantation of not only islets but also other organoids.

Characterization of the vascular heterogeneity within the pancreas has previously been lacking. Here, we develop strategies to enrich islet-specific endothelial cells (ISECs) and acinar-specific endothelial cells (ASECs) from three human pancreases and corroborate these findings with three published pancreatic datasets. Single-cell RNA sequencing reveals the unique molecular signatures of ISECs, including structural genes COL13A1, ESM1, PLVAP, UNC5B, and LAMA4, angiocrine genes KDR, THBS1, BMPs and CXCR4, and metabolic genes ACE, PASK and F2RL3. ASECs display distinct signatures including GPIHBP1, CCL14, CD74, AQP1, KLF4, and KLF2, which may manage the inflammatory and metabolic needs of the exocrine pancreas. Ligand-receptor analysis suggests ISECs and ASECs interact with LUM+ fibroblasts and RGS5+ pericytes and smooth muscle cells via VEGF-A:VEGFR2, CXCL12:CXCR4, and LIF:LIFR pathways. Comparative expression and immunohistochemistry indicate disruption of endothelial-expressed CD74, ESM1, PLVAP, THBD, VWA1, and VEGF-A cross-talk among vascular and other cell types in diabetes. Thus, our data provide a single-cell vascular atlas of human pancreas, enabling deeper understanding of pancreatic pathophysiology in health and disease. The pancreatic vasculature displays significant heterogeneity, with the islets perfused by a specialized microcirculation with greater density than the surrounding acinar tissue. Using single-cell RNA sequencing of human pancreases and integration with further data, the authors reveal the vascular transcriptomic heterogeneity in the healthy and diabetic pancreas.

Tissue-specific endothelial cells (ECs) regulate metabolism, inflammation, coagulation, organ development and regeneration. However, therapeutic application of EC transplantation requires scalable expansion of engraftable ECs that sustain their angiogenic and angiocrine functions. Here we identify a non-canonical aryl hydrocarbon receptor (AHR) pathway switched on by canonical AHR inhibitors that reactivates quiescent EC proliferation. Incubation of tissue-specific human ECs with AHR inhibitors, such as StemRegenin1 (SR1), increased EC proliferation by three-fold within an 8-day period. AHR inhibitors induced 100-fold greater expansion of 200,000 primary human adipose ECs to 2.4 × 1012 ECs, retaining in vivo vessel-forming and homeostatic functions in the recipient mice. AHR inhibitors induce a non-canonical AHR pathway by ornithine decarboxylase 1 (ODC1)-dependent synthesis of polyamines that drives EC cell cycle progression, detoxification of reactive oxygen species and oxidative phosphorylation metabolism, thereby recruiting hibernating ECs to accompany expanding EC populations without imposing replicative senescence. Therefore, AHR inhibitors, through transcriptional-independent protein–protein interactions, shepherd unrestricted human-scalable functional EC expansion, enabling cell therapies. Lin, Geng and colleagues identify a non-canonical AHR pathway that is activated by canonical AHR inhibitors, promoting the proliferation of quiescent endothelial cells with potential applications in cell therapy.

Myelodysplastic neoplasms (MDS) are genetically diverse hematopoietic cancers characterized by ineffective blood cell production, peripheral cytopenias, and an increased risk of acute myeloid leukemia. Diagnosis traditionally requires subjective histomorphologic assessment of a bone marrow biopsy sample. The potential biological and/or clinical relevance of subtle microarchitectural changes, unrecognizable using conventional methods, remains unknown. Here, we applied a recently developed AI-driven, whole slide imaging-based single-cell spatial proteomic profiling method to 77 annotated MDS and precursor state bone marrow tissue samples, including longitudinal cases. Compared to age-matched controls, MDS tissues showed significant changes in progenitor cell frequencies, morphologies of erythroid precursors and megakaryocytes, HSPC displacement from vasculature, abnormal progenitor cell clustering, and disrupted erythroid islands. Some alterations correlated more closely with specific mutations (e.g., SF3B1, TP53 ) than clinical risk scores (IPSS-M). Using all extracted tissue features, we developed a composite spatially informed "MDS severity score", which aligned with clinical and genetic parameters across serial samples. This work uncovers previously unrecognized, genotype-linked microarchitectural alterations in MDS, the measurement of which may enhance existing diagnostic and disease monitoring strategies.

Decoding the mechanisms governing the self-renewal of hematopoietic stem cells (HSCs) during their expansion in the fetal liver (FL) could unlock novel therapeutic strategies to expand transplantable HSCs, a long-standing challenge. To explore intrinsic and extrinsic regulation of FL-HSC self-renewal at single-cell resolution, we engineered a culture platform replicating the FL endothelial niche that supports the amplification of serially engraftable HSCs. Leveraging this platform together with single-cell index flow cytometry, live imaging, transplantation assays, and single-cell RNA sequencing, we demonstrate that differentiation latency, cell-division symmetry, and transcriptional signatures of biosynthetic dormancy are distinguishing properties of rare FL-HSCs capable of serial multilineage hematopoietic reconstitution. Our findings support a paradigm in which intrinsic programs and niche-derived signals together facilitate the symmetric self-renewal of FL-HSCs while delaying their active participation in hematopoiesis. Our study also provides a resource for future investigations into intrinsic and extrinsic signaling pathways governing FL-HSC self-renewal.

The human adenovirus serotype 5 E4ORF1 (Ad5E4ORF1) protein promotes primary endothelial cell survival and angiocrine functions by hijacking the cellular phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. However, the mechanism by which E4ORF1 activates PI3K in vascular cells remains largely unknown. Here, we show that Ad5E4ORF1 recruits multiple host scaffold proteins, including DLG1, which facilitates AKT activation in response to both Ad5E4ORF1 and endogenous receptor agonists in human endothelial cells. Furthermore, Ad5E4ORF1 specifically engages the human PI3K isoform p110α-p85β through multidomain interactions exclusively with p110α. Notably, E4ORF1 proteins from different adenoviral serotypes differentially interact with p110α, resulting in varying levels of AKT activation in endothelial cells. We propose that E4ORF1 specifically recognizes and allosterically activates p110α-p85β via direct multisite contacts with p110α.

Human intestinal organoids (HIOs) derived from human pluripotent stem cells co-differentiate both epithelial and mesenchymal lineages in vitro but lack important cell types such as neurons, endothelial cells, and smooth muscle, which limits translational potential. Here, we demonstrate that the intestinal stem cell niche factor, EPIREGULIN (EREG), enhances HIO differentiation with epithelium, mesenchyme, enteric neuroglial populations, endothelial cells, and organized smooth muscle in a single differentiation, without the need for co-culture. When transplanted into a murine host, HIOs mature and demonstrate enteric nervous system function, undergoing peristaltic-like contractions indicative of a functional neuromuscular unit. HIOs also form functional vasculature, demonstrated in vitro using microfluidic devices and in vivo following transplantation, where HIO endothelial cells anastomose with host vasculature. These complex HIOs represent a transformative tool for translational research in the human gut and can be used to interrogate complex diseases as well as for testing therapeutic interventions with high fidelity to human pathophysiology.

Transition between activation and quiescence states in hematopoietic stem and progenitor cells (HSPCs) is tightly governed by cell-intrinsic means and microenvironmental co-adaptation. Although this balance is fundamental for lifelong hematopoiesis and immunity, the underlying molecular mechanisms remain poorly defined. Multimodal analysis divulging differential transcriptional activity between distinct HSPC states indicates the presence of Fli-1 transcription factor binding motif in activated hematopoietic stem cells. We reveal that Fli-1 activity is essential during regenerative hematopoiesis in mice. Fli-1 directs activation programs while priming cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche through propagation of niche-derived angiocrine Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional hematopoietic stem cells impairments in the absence of Fli-1, without leukemic transformation. Applying FLI-1 transient modified-mRNA transduction into latent adult human mobilized HSPCs, enables their niche-mediated expansion and superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immunological regenerative medicine.