Biology subjects

Nakauchi, H.

Publications and source records attributed to Nakauchi, H..

At least 19 recordsLinked to original sources

bioRxiv2026

TorPause: Efficient Static Organ Preservation for Transplants

Transplantation is limited by how long organs survive outside the body. Existing preservation methods slow metabolism or sustain physiological support, but neither eliminates ischemia-reperfusion injury (IRI), which shortens storage and drives organ discard. Embryonic diapause and hibernation show that mammalian cells retain a latent capacity for reversible metabolic arrest. Screening the PI3K-mTOR axis, we identified a dual inhibitor that pauses embryonic stem cells reversibly at ambient oxygen and holds intact mouse embryos in arrest for 11 days. Combined with a mitochondria-targeted hydrogen sulfide donor, this yields TorPause: a two-component flush pairing metabolic pausing with mitochondrial protection, compatible with existing preservation protocols. In a rat liver transplantation model, TorPause reduced structural injury and improved recipient survival after severe warm ischemic storage, potentially expanding the usable donor pool.

developmental biology↗

bioRxiv2026

Hoxb5 Enriches Long-Term Hematopoietic Stem Cell Activity within the Mouse Fetal Liver Phenotypic HSC Compartment

Hematopoietic stem cells (HSCs) in the adult mouse can be prospectively isolated to near-purity through phenotypic markers, enabling detailed analysis of stem cell function. Homeobox B5 (Hoxb5) was previously identified as a definitive marker of long-term (LT) HSCs in adult bone marrow1. In contrast, fetal HSCs have not been purified to the same extent. Here, we show that Hoxb5 is expressed in fetal liver (FL) HSCs at embryonic day (E) 12.5-16.5 using a single-color tri-mCherry reporter driven by endogenous Hoxb5 regulation. Prospective purification by stringent multiparameter flow cytometry revealed Hoxb5 FL-HSCs to exhibit robust, multilineage reconstitution upon serial transplantation. Quantitative assays reveal that Hoxb5 enriches FL-HSCs to near-single-cell purity, analogous to its role in the adult bone marrow, underscoring its reliability in distinguishing LT-HSCs throughout hematopoietic ontogeny. Notably, Hoxb5 expression is not exclusive to FL-HSCs, as it is also detected across the fetal liver hematopoietic hierarchy and in fetal liver endothelial cells, suggesting developmental stage-specific regulation of its expression. In addition, single-cell RNA sequencing of FL-HSCs identified distinct transcriptional states defined by Hoxb5 expression. These findings establish Hoxb5 as a robust marker for enhancing the purification of fetal liver phenotypic HSCs (pHSC) and provide a framework for dissecting the molecular regulation of HSC ontogeny.

developmental biology↗

bioRxiv2026

Beta-Adrenergic Stimulation and MYH7 G256E Mutant Gene Dosage Drive Hypertrophic Cardiomyopathy Phenotype Penetrance

AimsHypertrophic cardiomyopathy (HCM) is the most prevalent genetic heart disorder, characterized by significant phenotypic variability even among individuals with identical MYH7 mutations. This study aims to elucidate factors contributing to this variability and identify drivers of phenotype penetrance. We compared the baseline phenotypes of a highly penetrant MYH7 H251N mutation and the variably penetrant MYH7 G256E mutation and investigated the impact of adding beta-adrenergic stimulation and homozygosity on disease phenotype penetrance using cardiomyocytes from an isogenic line of human induced pluripotent stem cells (hiPSC-CMs). Methods and ResultsIsogenic hiPSCs with MYH7 H251N and MYH7 G256E mutations were generated using CRISPR/Cas9 technology and differentiated into cardiomyocytes (CMs). Single-cell RNA sequencing (scRNAseq) and functional analysis of contractile function revealed consistent HCM phenotype presentation in H251N CMs, whereas G256E CMs exhibited a subtle and more variable phenotype. Beta-adrenergic stimulation induced a distinct metabolic stress response in G256E CMs, characterized by impaired mitochondrial ATP upregulation. Increasing mutant gene dosage from hetero- to homozygosity led to consistent increase in hypertrophic and structural gene expression changes in G256E CMs at RNA and protein levels. These changes were distinct from the changes observed with stress response. Importantly, homozygous G256E CMs exhibited a hypercontractile functional and disorganized structural phenotype. Across multiple experimental conditions, we identified consistent increase in cardiomyocyte specific transcriptomic markers such as NPPB, APOE, PDLIM3 and ANKRD1. ConclusionsOur study highlights the use of a variably penetrant MYH7 mutation to investigate factors that influence HCM phenotype penetrance. Specifically, we found that mutant gene dosage and beta-adrenergic stimulation induce distinct HCM disease phenotypes, providing novel insights into mechanisms that may contribute to variable disease expression in HCM. Translational PerspectiveHCM is characterized by significant phenotypic variability, complicating both diagnosis and clinical management. This study explores the factors driving HCM phenotype penetrance using isogenic hiPSC-CMs with MYH7 mutations. We demonstrate that beta-adrenergic stimulation and increased mutant gene dosage significantly impact HCM disease penetrance. Beta-adrenergic stimulation triggers metabolic stress responses, while increased gene dosage leads to a hypercontractile and structurally disorganized phenotype. These findings provide insight into how specific modifiers can shape disease-associated phenotypes in HCM model systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/729411v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@16a72d7org.highwire.dtl.DTLVardef@1999ca6org.highwire.dtl.DTLVardef@1fb8752org.highwire.dtl.DTLVardef@7148b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Xenophagocytosis blockade enhances interspecies chimerism

Organ shortage remains a major challenge in transplantation medicine. Interspecies blastocyst complementation is a promising approach to generate human organs in livestock hosts. However, getting xenogeneic donor cells to engraft and expand at early stages remains challenging. Here we identify an innate immune barrier, wherein host macrophages selectively recognize and eliminate viable xenogeneic donor cells. These events represent a form of phagoptosis and highlight a xenogeneic clearance process that we term xenophagocytosis. We identify the mechanism by which host macrophages selectively phagocytize xenogeneic donor cells: xenogeneic cells display elevated phosphatidylserine, an "eat-me" signal recognized by host macrophages through phagocytic receptor Axl. Xenophagocytosis blockade improves both rat and human donor chimerism in mouse embryos, indicating a conserved mechanism. These findings reveal potential mechanisms by which innate immune cells eliminate xenogeneic cells in early embryogenesis to preserve species integrity and offer improved strategies for generating human organs in livestock.

immunology↗

bioRxiv2025

Donor-Targeted Anti-HLA-A2 Antibody Shows Nanogram-Level Efficacy in a Novel GVHD Mouse Model

Acute graft-versus-host disease (GVHD) remains a life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), with limited treatment options for steroid-refractory cases. Current therapies broadly suppress immune responses, increasing infection risk and impairing graft function. We hypothesized that selective depletion of donor immune cells through HLA allele-specific targeting could offer a safer and more precise alternative. To test this, we developed a monoclonal antibody (AN7) against HLA-A2 by immunizing HLA-A24 transgenic mice with recombinant HLA-A2 tetramers, followed by hybridoma screening. AN7 specifically recognized HLA-A2 and HLA-A68, and was reformatted into murine IgG2a and human chimeric IgG1 recombinant antibodies. These retained binding specificity and mediated robust complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) against HLA-A2+ target cells. To evaluate therapeutic potential, we established a fully MHC-mismatched allo-HSCT model using HLA-A2 transgenic donor bone marrow in lethally irradiated BALB/c recipients. A single ultra-low dose of AN7 (1-100 ng) administered post-transplant significantly improved survival and reduced GVHD while preserving donor hematopoiesis. These findings highlight the potential of allele-specific antibody-mediated clearance as a mechanistically distinct approach to donor immune modulation in GVHD.

immunology↗

bioRxiv2025

The generation of viable, structurally integrated human-mouse chimaeras through enhanced hPSCs proliferation

The generation of human organs in animals through blastocyst complementation offers a promising solution to the shortage of transplantable organs. While human pluripotent stem cells (hPSCs) can contribute to interspecies chimaeric embryos when injected into preimplantation embryos of mice, pigs, or monkeys, their integration is often limited due to low chimaerism and segregation from host tissues, significantly impeding progress toward exogenic organ generation. Here, we demonstrate that co-overexpression of the anti-apoptotic gene BCL2 and the proto-oncogene MYCL, along with various cell cycle regulators significantly enhances human cell chimaerism by promoting cell proliferation. This strategy facilitates the generation of viable mouse pups containing hPSC-derived tissues without tumorigenesis. scRNA-seq analysis revealed that hPSCs already exit pluripotent by early post-implantation stage yet hPSCs with enhanced proliferation were able to integrate effectively into the cardiomyocytes and vasculature of both embryonic and extraembryonic tissues with gene expression profiles reflecting their structural integration. These findings highlight the critical role of cell cycle regulation in overcoming xenogeneic barriers. Our findings offer new insights into strategies for enhancing interspecies organogenesis and advancing the field of regenerative medicine. HighlightO_LIEnhancing hPSC proliferation increases human cell contribution in interspecies chimaeras C_LIO_LIAchieving the generation of viable human-mouse chimaeras without tumour formation C_LIO_LIhPSCs in post-implantation mouse epiblast exit pluripotency but retain the capacity to integrate into mouse embryogenesis C_LIO_LIhPSCs derivatives integrate into vasculature and cardiac tissues with lineage-matched transcriptional profiles C_LI

developmental biology↗

bioRxiv2025

A human-specific regulatory mechanism revealed in a preimplantation model

Stem cell-based human embryo models offer a unique opportunity for functional studies of the human-specific features of development. Here, we genetically and epigenetically manipulate human blastoids, a 3D embryo model of the blastocyst, to investigate the functional impact of HERVK LTR5Hs, a hominoid-specific endogenous retrovirus, on preimplantation development. We uncover a pervasive cis-regulatory contribution of LTR5Hs elements to the hominoid-specific diversification of the blastoids epiblast transcriptome. Many of the nearly 700 LTR5Hs genomic insertions in the human genome are unique to our own species. We show that at least one such human-specific LTR5Hs element is essential for the blastoid-forming potential via enhancing expression of the primate-specific ZNF729 gene, encoding a KRAB zinc finger protein. ZNF729 binds G/C-rich sequences, extremely abundant at gene promoters associated with basic cellular functions, such cell proliferation and metabolism. Surprisingly, despite mediating recruitment of TRIM28, at many of these promoters ZNF729 acts as a transcriptional activator. Together, our results illustrate how recently emerged transposable elements and genes can confer developmentally essential functions in humans.

developmental biology↗

bioRxiv2025

Generation of iPSC-derived CD4+ Th1 cells enhancing chimeric antigen receptor-T cell cytotoxicity

CD4+ T cells are anticipated to enhance the overall immune response, including the anti-tumor activity of chimeric antigen receptor (CAR)-T cell therapy. In the past, we established a culture system to generate CD8+ T cells from iPS cells (iPSCs); however, it was challenging to generate CD4+ T cells. Drawing inspiration from the observation that adult T cell leukemia (ATL) cells are consistently CD4+ and possess Treg characteristics, we successfully generated CD4+ Treg cells by reprogramming ATL cells into iPSCs and then differentiating them into T cells. Gene expression analysis of this generation system suggested that RUNX3 serves as a key regulator in T cell differentiation within the ex vivo generation system. By knocking out RUNX3, we demonstrated the generation of antigen-specific CD4+ Th1 cells via the iPSC route, thereby enhancing the activity of CD8+ CAR-T cells against GD2-expressing lymphoma. These technologies hold significant promise for contributing to "off-the-shelf" immunotherapies against malignant tumors, including solid tumors.

immunology↗

bioRxiv2025

Development of iPSC-Derived T Cells Targeting EGFR Neoantigens in Non-Small Cell Lung Cancer

A long-sought goal of cancer immunotherapy is to mass-produce T cells that specifically target tumor neoantigens. One decisive challenge is the identification of neoantigens derived from cancer driver genes. Here, we identify T cells that recognize the NSCLC-associated EGFR C797S mutation, which confers resistance to current inhibitors and is linked to poor prognosis. To overcome limitations in T cell availability, we reprogrammed EGFR C797S-specific T cells into induced pluripotent stem cells (iPSCs) and re-differentiated them into CD8 T cells. These iPSC-derived T cells specifically recognized the EGFR C797S mutation and effectively killed cancer cells expressing this mutation. Our findings underscore the potential of targeting driver mutation-derived neoantigens for immunotherapy and demonstrate that iPSC-derived T cells can mediate antitumor effects. Collectively, this approach combining neoantigen identification with T cell reprogramming may offer a promising strategy for targeting drug-resistant tumors.

immunology↗

bioRxiv2024

DNMT3AR882H Is Not Required for Disease Maintenance in Primary Human AML, but Is Associated With Increased Leukemia Stem Cell Frequency

Genetic mutations are being thoroughly mapped in human cancers, yet a fundamental question in cancer biology is whether such mutations are functionally required for cancer initiation, maintenance of established cancer, or both. Here, we study this question in the context of human acute myeloid leukemia (AML), where DNMT3AR882 missense mutations often arise early, in pre-leukemic clonal hematopoiesis, and corrupt the DNA methylation landscape to initiate leukemia. We developed CRISPR-based methods to directly correct DNMT3AR882 mutations in leukemic cells obtained from patients. Surprisingly, DNMT3AR882 mutations were largely dispensable for disease maintenance. Replacing DNMT3AR882 mutants with wild-type DNMT3A did not impair the ability of AML cells to engraft in vivo, and minimally altered DNA methylation. Taken together, DNMT3AR882 mutations are initially necessary for AML initiation, but are largely dispensable for disease maintenance. The notion that initiating oncogenes differ from those that maintain cancer has important implications for cancer evolution and therapy. STATEMENT OF SIGNIFICANCEUnderstanding which driver mutations are required for cancer initiation, maintenance, or both phases remains poorly understood. Here, we uncover that highly prevalent pre-leukemic DNMT3A mutations are only required during disease initiation, but become dispensable after leukemic transformation, uncovering the context-specific role of this driver mutation with important therapeutic implications.

cancer biology↗

bioRxiv2024

Inter-cellular mRNA Transfer Alters Human Pluripotent Stem Cell State

Inter-cellular transmission of mRNA is being explored in mammalian species using immortal cell lines (1-3). Here, we uncover an inter-cellular mRNA transfer phenomenon that allows for the adaptation and reprogramming of human primed pluripotent stem cells (hPSCs). This process is induced by the direct cell contact-mediated coculture with mouse embryonic stem cells (mESCs) under the condition impermissible for human primed PSC culture. Mouse-derived mRNA contents are transmitted into adapted hPSCs only in the coculture. Transfer-specific mRNA analysis show the enrichment for divergent biological pathways involving transcription/translational machinery and stress-coping mechanisms, wherein such transfer is diminished when direct cell contacts are lost. After 5 days of mESC culture, surface marker analysis, and global gene profiling confirmed that mRNA transfer-prone hPSC efficiently gains a naive-like state. Furthermore, transfer-specific knockdown experiments targeting mouse-specific transcription factor-coding mRNAs in hPSC show that mouse-derived Tfcp2l1, Tfap2c, and Klf4 are indispensable for human naive-like conversion. Thus, inter-species mRNA transfer triggers cellular reprogramming in mammalian cells. Our results support that episodic mRNA transfer can occur in cell cooperative and competitive processes(4), which provides a fresh perspective on understanding the roles of mRNA mobility for intra- and inter-species cellular communications.

cell biology↗

bioRxiv2024

Stable platelet production via the bypass pathway explains the long-term reconstitution capacity of hematopoietic stem cells

Precise understanding how hematopoietic stem cells (HSCs) differentiate in vivo is difficult because we can not trace the in vivo differentiation of HSCs. The single-cell transplantation assay and our own paired daughter cell assay of phenotypic HSCs has revealed the presence of HSCs with reconstitution capacity whose differentiation potential is restricted to the myeloid lineage (MySCs) and the presence of novel direct differentiation pathway form HSCs to MySCs (named myeloid bypass pathway). However, how HSCs differentiate in vivo during hematopoiesis has remained unclear since the paired daughter cell assay was performed partially ex vivo. Aiming to characterize HSCs, including the myeloid bypass pathway, we examined the kinetics of HSC differentiation using a mathematical model. We analyzed data from single-cell transplantation assays in which five blood cell lineages were followed successively after transplantation. An age-related skewing to the myeloid lineage was quantitatively indicated as the production of B cells reduced with age. Dependence on platelet bypass increased with aging and consistently high dependence was associated with long-term reconstitution capacity of the HSCs. Focusing on the ratio of chimerism among cell lineages, the characteristics of dependence on platelet bypass can be accurately determined by the ratio of erythrocyte to platelet chimerism at 8 weeks after transplantation. This new identification criterion is an indicator of long-term reconstitution of HSCs that does not rely on observation of long-term transplantation experiments. These findings reveal the novel characteristics of HSCs related to aging and stemness and highlight the importance of the bypass pathway in HSC differentiation. Significance statementIn vivo differentiation of hematopoietic stem cells (HSCs) is important for understanding blood cell production. Here we investigated the differentiation kinetics of HSCs in single-cell transplantation assays by using a mathematical model. Our findings demonstrate the importance of the bypass pathway in platelet production for the long-term reconstitution capacity of HSCs. These results also suggest the utility of studying time changes in HSC differentiation. The new HSC characteristics and its detection criteria identified in this study are expected to be useful in understanding HSC diversity and aging.

cell biology↗

bioRxiv2024

Intraplacental injection of human iPSC-derived PDX1+ pancreatic progenitors prolongs Pdx1-deficient mice survival

Interspecies chimeras comprising human tissues have potential for use in disease modeling and regenerative medicine. Here, we successfully transplanted human induced pluripotent stem cell (iPSC)-derived PDX1+ pancreatic progenitor cells into Pdx1-deficient mouse embryos via intraplacental injection. The engrafted human cells predominantly localized to the duodenum, produced insulin, and extended the lifespan of Pdx1-/- mice by up to 10 days after birth. Transcriptomic analyses confirmed human pancreatic gene expression in human cells engrafted into the mouse duodenum. Our findings demonstrated the feasibility of generating interspecies chimeras with functional human pancreatic cells through in utero transplantation of lineage-committed progenitors. This approach circumvents developmental barriers while minimizing ethical concerns associated with PSCs. However, the incomplete rescue of the Pdx1-/- phenotype highlights the need for further research to enhance human cell engraftment and tissue integration. Overall, this study provides a foundation for developing human-animal chimera models for studying human development and regenerative therapies.

cell biology↗

bioRxiv2024

Disentangling cell-intrinsic and extrinsic factors underlying gene expression evolution

A key goal of developmental biology is to determine the extent to which cells and organs develop autonomously, as opposed to requiring interactions with other cells or environmental factors. Chimeras have played a foundational role in this by enabling qualitative classification of cell-intrinsically vs. extrinsically driven processes. Here, we extend this framework to precisely decompose evolutionary divergence in any quantitative trait into cell-intrinsic, extrinsic, and intrinsic-extrinsic interaction components. Applying this framework to thousands of gene expression levels in reciprocal rat-mouse chimeras, we found that the majority of their divergence is attributable to cell-intrinsic factors, though extrinsic factors also play an integral role. For example, a rat-like extracellular environment extrinsically up-regulates the expression of a key transcriptional regulator of the endoplasmic reticulum (ER) stress response in some but not all cell types, which in turn strongly predicts extrinsic up-regulation of its target genes and of the ER stress response pathway as a whole. This effect is also seen at the protein level, suggesting propagation through multiple regulatory levels. Applying our framework to a cellular trait, neuronal differentiation, revealed a complex interaction of intrinsic and extrinsic factors. Finally, we show that imprinted genes are dramatically mis-expressed in species-mismatched environments, suggesting that mismatch between rapidly evolving intrinsic and extrinsic mechanisms controlling gene imprinting may contribute to barriers to interspecies chimerism. Overall, our conceptual framework opens new avenues to investigate the mechanistic basis of developmental processes and evolutionary divergence across myriad quantitative traits in any multicellular organism.

evolutionary biology↗

bioRxiv2024

Secreted Particle Information Transfer (SPIT) - A Cellular Platform For In Vivo Genetic Engineering

A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for in vivo genetic engineering, Secreted Particle Information Transfer (SPIT) that utilizes human cells as delivery vectors for in vivo genetic engineering. We demonstrate the application of SPIT for cell-cell delivery of Cre recombinase and CRISPR-Cas9 enzymes, we show that genetic logic can be incorporated into SPIT and present the first demonstration of human cells as a delivery platform for in vivo genetic engineering in immunocompetent mice. We successfully applied SPIT to genetically modify multiple organs and tissue stem cells in vivo including the liver, spleen, intestines, peripheral blood, and bone marrow. We anticipate that by harnessing the large packaging capacity of a human cells nucleus, the ability of human cells to engraft into patients long term and the capacity of human cells for complex genetic programming, that SPIT will become a paradigm shifting approach for in vivo genetic engineering.

synthetic biology↗

bioRxiv2022

Large-scale in vivo CRISPR screens identify SAGA complex members as a key regulators of HSC lineage commitment and aging

The biological mechanisms that sustain the vast blood production required for healthy life remain incompletely understood. To search for novel regulators of hematopoiesis, we performed genome-wide in vivo hematopoietic stem cell (HSC)-based CRISPR knockout screens for regulators of hematopoiesis. We discovered SAGA complex members, including Tada2b and Taf5l, as key regulators of hematopoiesis. Loss of Tada2b or Taf5l strongly inhibited hematopoiesis in vivo, led to a buildup of immature hematopoietic cells in the bone marrow, and was associated with upregulation of interferon pathway genes. Loss of these factors also enhanced the cell outgrowth and the interferon pathway in an in vivo human myelodysplastic syndrome model, suggesting that loss of SAGA complex activity could contribute to hematological disease progression. In summary, this study has identified the SAGA complex as an important regulator of hematopoiesis.

genetics↗

bioRxiv2021

A highly efficient reporter system for identifying and characterizing in vitro expanded hematopoietic stem cells

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells being non-HSCs and single cell-initiated cultures displaying substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. Linking single clone functional transplantation data with single clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to actively cycling fetal liver HSCs and shares a gene expression signature with functional HSCs from all sources, including Prdm16, Fstl1 and Palld. This new tool can now be applied to a wide-range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

cell biology↗

bioRxiv2020

Streamlined and quantitative detection of chimerism in mouse tissue using digital PCR

Animal chimeras are widely used for biomedical discoveries, from developmental biology to cancer research. However, the accurate quantitation of mixed cell types in chimeric and mosaic tissues has challenges. Here, we have developed and characterized a droplet digital PCR single-nucleotide discrimination assay to detect chimerism among albino and non-albino mouse strains. In addition, we have validated that this assay is compatible with crude lysate from most organs, drastically streamlining sample preparation. This chimerism detection assay has many additional advantages over existing methods including its robust nature, minimal technical bias, and ability to report the total number of cells in a prepared sample. Importantly, the concepts developed and discussed here are readily adapted to other genomic loci to accurately measure mixed cell populations in any tissue.

genetics↗