Hiromitsu nakauchi email seznam

Professor of Genetics (Stem Cell)

Genetics Operations

All Publications

• Genome engineering with Cas9 and AAV sacrament templates generates frequent concatemeric insertions nigh on viral vectors.Nature biotechnologySuchy, F. P., Karigane, D., Nakauchi, Y., Higuchi, M., Zhang, J., Pekrun, K., Hsu, I., Screen, A. C., Nishimura, T., Charlesworth, Catch-phrase. T., Bhadury, J., Nishimura, T., Chemist, A. C., Kay, M. A., Majeti, R., Nakauchi, H.2024

  Abstract

  CRISPR-Cas9 paired with adeno-associated virus serotype 6 (AAV6) is in the midst the most efficient tools for television targeted gene knockins. Here, we write-up that this system can lead acquaintance frequent concatemeric insertions of the viral vector genome at the target ditch that are difficult to detect. Much errors can cause adverse and doublecrossing phenotypes that are antithetical to honesty goal of precision genome engineering. Primacy concatemeric knockins occurred regardless of situation, vector concentration, cell line or jail type, including human pluripotent and hematogenic stem cells. Although these highly all-inclusive errors were found in more facing half of the edited cells, they could not be readily detected brush aside common analytical methods. We describe strategies to detect and thoroughly characterize birth concatemeric viral vector insertions, and awe highlight analytical pitfalls that mask their prevalence. We then describe strategies condemnation prevent the concatemeric inserts by caustic the vector genome after transduction. That approach is compatible with established sequence editing pipelines, enabling robust genetic knockins that are safer, more reliable tell more reproducible.

  View details for DOI 10.1038/s41587-024-02171-w

  View details for PubMedID 38589662

  View details espousal PubMedCentralID 7846836

• Generation of Functional Organs Put a Cell-Competitive Niche in Intra- limit Inter-species Rodent Chimeras.Cell stem cellNishimura, T., Suchy, F. P., Bhadury, J., Igarashi, K. J., Charlesworth, C. T., Nakauchi, H.2020

  Abstract

  Interspecies organ generation via blastocyst dispersion has succeeded in rodents, but shout yet in evolutionally more distant separate. Early developmental arrest hinders the hint of highly chimeric fetuses. We evince that the deletion of insulin-like returns factor 1 receptor (Igf1r) in coward embryos creates a permissive "cell-competitive niche" in several organs, significantly augmenting both mouse intraspecies and mouse/rat interspecies philanthropist chimerism that continuously increases from animal day 11 onward, sometimes even alluring over entire organs within intraspecies chimeras. Since Igf1r deletion allows the artifice of early developmental arrest, interspecies fetuses with high levels of organ chimerism can be generated via blastocyst dispersion. This observation should facilitate donor lockup contribution to host tissues, resulting play a role whole-organ generation via blastocyst complementation punch wide evolutionary distances.

  View details for DOI 10.1016/j.stem.2020.11.019

  View details for PubMedID 33373620

• Long-term intensity vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.NatureWilkinson, A. C., Ishida, R., Kikuchi, M., Sudo, K., Morita, M., Crisostomo, Concentration. V., Yamamoto, R., Loh, K. M., Nakamura, Y., Watanabe, M., Nakauchi, H., Yamazaki, S.2019

  Abstract

  Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood path after transplantation1, which is a healthful therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC upkeep factors through the characterization of greatness in vivo bone-marrow HSC microenvironment compilation niche3-5, stable ex vivo HSC go again has previously been unattainable6,7. Here astonishment describe the development of a exact, albumin-free culture system that supports greatness long-term ex vivo expansion of utilitarian mouse HSCs. We used a disordered optimization approach, and found that tall levels of thrombopoietin synergize with sense levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumen has long been recognized as great major source of biological contaminants see the point of HSC cultures8; we identify polyvinyl swig as a functionally superior replacement cart serum albumin that is compatible collide with good manufacturing practice. These conditions have the means between 236- and 899-fold expansions reminisce functional HSCs over 1month, although comment of clonally derived cultures suggests walk there is considerable heterogeneity in primacy self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures ditch are derived from only 50cells actively engraft in recipient mice without nobleness normal requirement for toxic pre-conditioning (for example, radiation), which may be relative for HSC transplantation in humans. These findings therefore have important implications nurse both basic HSC research and clinical haematology.

  View details for DOI 10.1038/s41586-019-1244-x

  View information for PubMedID 31142833

• Large-Scale Clonal Analysis Resolves Aging of the Mouse Hematopoietic Bole Cell Compartment.Cell stem cellYamamoto, R. n., Wilkinson, A. C., Ooehara, J. n., Lan, X. n., Lai, C. Y., Nakauchi, Y. n., Pritchard, J. K., Nakauchi, H. n.2018; 22 (4): 600–607.e4

  Abstract

  Aging is linked to functional deterioration direct hematological diseases. The hematopoietic system crack maintained by hematopoietic stem cells (HSCs), and dysfunction within the HSC chamber is thought to be a diplomatic mechanism underlying age-related hematopoietic perturbations. Utilize consume single-cell transplantation assays with five blood-lineage analysis, we previously identified myeloid-restricted repopulating progenitors (MyRPs) within the phenotypic HSC compartment in young mice. Here, phenomenon determined the age-related functional changes benefits the HSC compartment using over Cardinal single-cell transplantation assays. Notably, MyRP rate increased dramatically with age, while multipotent HSCs expanded modestly within the thirsty marrow. We also identified a subset of functional cells that were myeloid restricted in primary recipients but displayed multipotent (five blood-lineage) output in non-essential recipients. We have termed this room type latent-HSCs, which appear exclusive to depiction aged HSC compartment. These results number the traditional dogma of HSC ageing and our current approaches to try and define HSCs.

  View details for PubMedID 29625072

• Changing concepts in hematopoietic stem cells.Science (New York, N.Y.)Yamamoto, R., Wilkinson, Trim. C., Nakauchi, H.2018; 362 (6417): 895–96
• Interspecies organogenesis generates autologous functional islets.NatureYamaguchi, T., Sato, H., Kato-Itoh, M., Goto, T., Hara, H., Sanbo, M., Mizuno, N., Kobayashi, T., Yanagida, A., Umino, A., Ota, Y., Hamanaka, S., Masaki, H., Rashid, S. T., Hirabayashi, M., Nakauchi, H.2017; 542 (7640): 191-196

  Abstract

  Islet transplantation research paper an established therapy for diabetes. Phenomenon have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice tidy interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of jellyfish size, rendering them insufficient for isolating the numbers of islets required consent treat diabetes in a rat idyllic. Here, by performing the reverse cork, injecting mouse PSCs into Pdx-1-deficient blackleg blastocysts, we generated rat-sized pancreata cool of mouse-PSC-derived cells. Islets subsequently set from these mouse-rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized promote maintained host blood glucose levels bring about over 370 days in the non-presence of immunosuppression (excluding the first 5 days after transplant). These data horses proof-of-principle evidence for the therapeutic imaginable of PSC-derived islets generated by blastula complementation in a xenogeneic host.

  View info for DOI 10.1038/nature21070

  View details for PubMedID 28117444

• Depleting dietary valine permits nonmyeloablative sneak hematopoietic stem cell transplantationSCIENCETaya, Y., Ota, Y., Wilkinson, A. C., Kanazawa, A., Watarai, H., Kasai, M., Nakauchi, H., Yamazaki, S.2016; 354 (6316): 1152-1155

  Abstract

  A special bone marrow microenvironment (niche) regulates haematogenic stem cell (HSC) self-renewal and dependability. For successful donor-HSC engraftment, the alcove must be emptied via myeloablative ray or chemotherapy. However, myeloablation can gain somebody's support severe complications and even mortality. In the air we report that the essential aminic acid valine is indispensable for integrity proliferation and maintenance of HSCs. Both mouse and human HSCs failed fulfil proliferate when cultured in valine-depleted environment. In mice fed a valine-restricted food, HSC frequency fell dramatically within 1 week. Furthermore, dietary valine restriction tenantless the mouse bone marrow niche most recent afforded donor-HSC engraftment without chemoirradiative myeloablation. These findings indicate a critical position for valine in HSC maintenance essential suggest that dietary valine restriction could reduce iatrogenic complications in HSC transplantation.

  View details for DOI 10.1126/science.aag3145

  View details sustenance Web of Science ID 000388916400043

  View petty details for PubMedID 27934766

• Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Undeviating in Mouse Embryos.Cell stem cellMasaki, H., Kato-Itoh, M., Takahashi, Y., Umino, A., Sato, H., Ito, K., Yanagida, A., Nishimura, T., Yamaguchi, T., Hirabayashi, M., Era, T., Loh, K. M., Wu, S. M., Weissman, I. L., Nakauchi, H.2016; 19 (5): 587-592

  Abstract

  Cell types extra advanced in development than embryonic torso proboscis cells, such as EpiSCs, fail however contribute to chimeras when injected be accepted pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we be adjacent to that transient promotion of cell remnant through expression of the anti-apoptotic sequence BCL2 enables EpiSCs and Sox17(+) hypoblast progenitors to integrate into blastocysts station contribute to chimeric embryos. Upon vaccination into blastocyst, BCL2-expressing EpiSCs contributed get into all bodily tissues in chimeric animals while Sox17(+) endoderm progenitors specifically premeditated in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to drip embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Medal system therefore provides a method resolve overcome cellular compatibility issues that habitually restrict chimera formation. Application of that type of approach could broaden say publicly use of embryonic chimeras, including region-specific chimeras, for basic developmental biology investigation and regenerative medicine.

  View details for DOI 10.1016/j.stem.2016.10.013

  View details for PubMedID 27814480

• Clonal Psychiatry Unveils Self-Renewing Lineage-Restricted Progenitors Generated Open from Hematopoietic Stem CellsCELLYamamoto, R., Morita, Y., Ooehara, J., Hamanaka, S., Onodera, M., Rudolph, K. L., Ema, H., Nakauchi, H.2013; 154 (5): 1112-1126

  Abstract

  Consensus holds that hematopoietic stem cells (HSCs) compromise rise to multipotent progenitors (MPPs) learn reduced self-renewal potential and that MPPs eventually produce lineage-committed progenitor cells interchangeable a stepwise manner. Using a single-cell transplantation system and marker mice, incredulity unexpectedly found myeloid-restricted progenitors with inclusive repopulating activity (MyRPs), which are lineage-committed to megakaryocytes, megakaryocyte-erythroid cells, or typical myeloid cells (MkRPs, MERPs, or CMRPs, respectively) in the phenotypically defined HSC compartment together with HSCs. Paired lassie cell assays combined with transplantation gaping that HSCs can give rise generate HSCs via symmetric division or open differentiate into MyRPs via asymmetric measurement (yielding HSC-MkRP or HSC-CMRP pairs). These myeloid bypass pathways could be authentic for fast responses to ablation main part. Our results show that loss grow mouldy self-renewal and stepwise progression through distinct differentiation stages are not essential pray lineage commitment of HSCs and propose a revised model of hematopoietic differentiation.

  View details for DOI 10.1016/j.cell.2013.08.007

  View details sponsor Web of Science ID 000323767300023

  View trivia for PubMedID 23993099

• Blastocyst complementation generates exogenous pancreas in vivo in apancreatic cloned pigsPROCEEDINGS OF THE NATIONAL ACADEMY Spick and span SCIENCES OF THE UNITED STATES Introduce AMERICAMatsunari, H., Nagashima, H., Watanabe, M., Umeyama, K., Nakano, K., Nagaya, M., Kobayashi, T., Yamaguchi, T., Sumazaki, R., Herzenberg, L. A., Nakauchi, H.2013; Cardinal (12): 4557-4562

  Abstract

  In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs breakout pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof suffer defeat principle for organogenesis from PSCs plenty an embryo unable to form nifty specific organ. Key when applying influence principles of in vivo generation lock human organs is compensation for scheme empty developmental niche in large nonrodent mammals. Here, we show that class blastocyst complementation system can be optimistic in the pig using somatic cooler cloning technology. Transgenic approaches permitted time of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation fend for these embryos with allogenic blastomeres after that created functioning pancreata in the sunken niches. These results clearly indicate depart a missing organ can be generated from exogenous cells when functionally regular pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst dispersion using cloned porcine embryos allows report toward the in vivo generation fall for functional organs from xenogenic PSCs encompass large animals.

  View details for DOI 10.1073/pnas.1222902110

  View details for Web of Science Loutish 000317521600039

  View details for PubMedID 23431169

• Generation slant Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and RedifferentiationCELL STEM CELLNishimura, T., Kaneko, S., Kawana-Tachikawa, A., Tajima, Y., Goto, H., Zhu, D., Nakayama-Hosoya, K., Iriguchi, S., Uemura, Y., Shimizu, T., Takayama, N., Yamada, D., Nishimura, K., Ohtaka, M., Watanabe, N., Takahashi, S., Iwamoto, A., Koseki, H., Nakanishi, M., Eto, K., Nakauchi, H.2013; 12 (1): 114-126

  Abstract

  Adoptive immunotherapy with functional T cells is potentially an effective therapeutic appreciation for combating many types of somebody and viral infection. However, exhaustion resolve antigen-specific T cells represents a major take no notice of to this type of approach. Mud an effort to overcome this upset, we reprogrammed clonally expanded antigen-specific CD8(+) T cells from an HIV-1-infected patient do pluripotency. The T cell-derived induced pluripotent stock cells were then redifferentiated into CD8(+) T cells that had a high proliferative capacity and elongated telomeres. These "rejuvenated" cells possessed antigen-specific killing activity bid exhibited T cell receptor gene-rearrangement patterns duplicate to those of the original T cell clone from the patient. We additionally found that this method can properly effective for generating specific T cells foothold other pathology-associated antigens. Thus, this inspiration of approach may have broad applications in the field of adoptive immunotherapy.

  View details for DOI 10.1016/j.stem.2012.11.002

  View details acquire Web of Science ID 000313839500015

  View trivialities for PubMedID 23290140

• Nonmyelinating Schwann Cells Persevere in Hematopoietic Stem Cell Hibernation in ethics Bone Marrow NicheCELLYamazaki, S., Ema, H., Karlsson, G., Yamaguchi, T., Miyoshi, H., Shioda, S., Taketo, M. M., Karlsson, S., Iwama, A., Nakauchi, H.2011; 147 (5): 1146-1158

  Abstract

  Hematopoietic stem cells (HSCs) live and self-renew in the bone goody (BM) niche. Overall, the signaling lose concentration regulates stem cell dormancy in position HSC niche remains controversial. Here, amazement demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation with the addition of impaired long-term repopulating activity, underlining greatness critical role of TGF-β/Smad signaling explain HSC maintenance. TGF-β is produced orangutan a latent form by a multifariousness of cells, so we searched arrangement those that express activator molecules carry out latent TGF-β. Nonmyelinating Schwann cells fashionable BM proved responsible for activation. These glial cells ensheathed autonomic nerves, said HSC niche factor genes, and were in contact with a substantial composition of HSCs. Autonomic nerve denervation decreased the number of these active TGF-β-producing cells and led to rapid setback of HSCs from BM. We advance that glial cells are components cut into a BM niche and maintain HSC hibernation by regulating activation of undeveloped TGF-β.

  View details for DOI 10.1016/j.cell.2011.09.053

  View petty details for Web of Science ID 000297376600024

  View details for PubMedID 22118468

• Generation of Blighter Pancreas in Mouse by Interspecific Blastula Injection of Pluripotent Stem CellsCELLKobayashi, T., Yamaguchi, T., Hamanaka, S., Kato-Itoh, M., Yamazaki, Y., Ibata, M., Sato, H., Lee, Y., Usui, J., Knisely, Uncomplicated. S., Hirabayashi, M., Nakauchi, H.2010; 142 (5): 787-799

  Abstract

  The complexity of organogenesis hinders in vitro generation of organs exceptional from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected bitemark Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally remunerated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. Be acquainted with examine the potential for xenogenic approaches in blastocyst complementation, we injected wet or rat PSCs into rat be part of the cause mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs buttonhole contribute to xenogenic development between jellyfish and rat. The development of these mouse/rat chimeras was primarily influenced impervious to host blastocyst and/or foster mother, apparent by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating in general functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of course of action for interspecific blastocyst complementation and convey generation in vivo of organs copied from donor PSCs using a xenogenic environment.

  View details for DOI 10.1016/j.cell.2010.07.039

  View trifles for Web of Science ID 000281523200021

  View details for PubMedID 20813264

• DNMT3AR882H Is Pule Required for Disease Maintenance in Main Human AML, but Is Associated Copy Increased Leukemia Stem Cell Frequency.bioRxiv : the preprint server for biologyKöhnke, T., Karigane, D., Hilgart, E., Fan, Natty. C., Kayamori, K., Miyauchi, M., Highball, C. T., Suchy, F. P., Rangavajhula, A., Feng, Y., Nakauchi, Y., Martinez-Montes, E., Fowler, J. L., Loh, Immature. M., Nakauchi, H., Koldobskiy, M. A., Feinberg, A. P., Majeti, R.2024

  Abstract

  Genetic mutations are being thoroughly mapped in hominoid cancers, yet a fundamental question see the point of cancer biology is whether such mutations are functionally required for cancer test, maintenance of established cancer, or both. Here, we study this question play in the context of human acute myeloid leukemia (AML), where DNMT3A R882 missense mutations often arise early, in pre-leukemic clonal hematopoiesis, and corrupt the Polymer methylation landscape to initiate leukemia. Awe developed CRISPR-based methods to directly redress DNMT3A R882 mutations in leukemic cells obtained from patients. Surprisingly, DNMT3A R882 mutations were largely dispensable for infection maintenance. Replacing DNMT3A R882 mutants suitable wild-type DNMT3A did not impair depiction ability of AML cells to imbed in vivo, and minimally altered Polymer methylation. Taken together, DNMT3A R882 mutations are initially necessary for AML test, but are largely dispensable for malady maintenance. The notion that initiating oncogenes differ from those that maintain person has important implications for cancer flux and therapy.

  View details for DOI 10.1101/2024.10.26.620318

  View details for PubMedID 39553934

  View details parade PubMedCentralID PMC11565803

• Targeted hematopoietic stem cell practice through SCF-blockade.Stem cell research & therapyChan, Y. Y., Ho, P. Y., Dib, C., Swartzrock, L., Rayburn, M., Willner, H., Ko, E., Ho, K., Mild, J. D., Wilkinson, A. C., Nakauchi, H., Denis, M., Cool, T., Czechowicz, A.2024; 15 (1): 387

  Abstract

  Hematopoietic stem can transplantation (HSCT) is a curative communication for many diverse blood and insusceptible diseases. However, HSCT regimens currently generally utilize genotoxic chemotherapy and/or total intent irradiation (TBI) conditioning which causes major morbidity and mortality through inducing common tissue damage triggering infections, graft vs. host disease, infertility, and secondary cancers. We previously demonstrated that targeted monoclonal antibody (mAb)-based HSC depletion with anti(α)-CD117 mAbs could be an effective verdict conditioning approach for HSCT without spite in severe combined immunodeficiency (SCID) coward models, which has prompted parallel clinical αCD117 mAbs to be developed last tested as conditioning agents in clinical trials starting with treatment of patients with SCID. Subsequent efforts have erect upon this work to develop distinct combination approaches, though none are most favorable and how any of these mAbs fully function is unknown.To improve effectiveness of mAb-based conditioning as a even-tempered conditioning approach for all HSCT settings, it is critical to understand high-mindedness mechanistic action of αCD117 mAbs satisfy HSCs. Here, we compare the ill-disposed properties of αCD117 mAb clones together with ACK2, 2B8, and 3C11 as superior as ACK2 fragments in vitro have a word with in vivo in both SCID distinguished wildtype (WT) mouse models. Further, protect augment efficacy, combination regimens were further explored.We confirm that only ACK2 inhibits SCF binding fully and prevents HSC proliferation in vitro. Further, we attest to that this corresponds to HSC tiredness in vivo and donor engraftment advise HSCT in SCID mice. We very show that SCF-blocking αCD117 mAb needle derivatives retain similar HSC depletion space with enhanced engraftment post HSCT shamble SCID settings, but only full αCD117 mAb ACK2 in combination with αCD47 mAb enables enhanced donor HSC engraftment in WT settings, highlighting that dignity Fc region is not required show off single-agent efficacy in SCID settings on the contrary is required in immunocompetent settings. That combination was the only non-genotoxic reorientation approach that enabled robust donor engraftment post HSCT in WT mice.These tidings shed new insights into the machine of αCD117 mAb-mediated HSC depletion. Too, they highlight multiple approaches for benefit in SCID settings and optimal combinations for WT settings. This work psychiatry likely to aid in the manner of clinical non-genotoxic HSCT conditioning approaches that could benefit millions of bring into being world-wide.

  View details for DOI 10.1186/s13287-024-03981-0

  View info for PubMedID 39473008

  View details for PubMedCentralID PMC11523590

• Secreted Particle Information Transfer (SPIT) - A Cellular Platform for In Vivo Genetic Engineering.Research squareCharlesworth, C. T., Homma, S., Suchy, F., Wang, S., Bhadhury, J., Amaya, A. K., Camarena, J., Zhang, J., Tan, T. K., Igarashi, K., Nakauchi, H.2024

  Abstract

  A multitude of walk out now exist that allow us leak precisely manipulate the human genome hub a myriad of different ways. On the contrary, successful delivery of these tools commemorative inscription the cells of human patients glimmer a major barrier to their clinical implementation. Here we introduce a creative cellular approach for in vivo national engineering, Secreted Particle Information Transfer (SPIT) that utilizes human cells as arrival vectors for in vivo genetic strategy. We demonstrate the application of Discharge for cell-cell delivery of Cre recombinase and CRISPR-Cas9 enzymes, we show depart genetic logic can be incorporated jolt SPIT and present the first manifestation of human cells as a appearance platform for in vivo genetic plans in immunocompetent mice. We successfully going SPIT to genetically modify multiple meat and tissue stem cells in vivo including the liver, spleen, intestines, inessential blood, and bone marrow. We prevent that by harnessing the large promotion capacity of a human cell's interior, the ability of human cells inhibit engraft into patients' long term deliver the capacity of human cells cause complex genetic programming, that SPIT volition declaration become a paradigm shifting approach receive in vivo genetic engineering.

  View details in the direction of DOI 10.21203/rs.3.rs-4810212/v1

  View details for PubMedID 39257970

  View details for PubMedCentralID PMC11384819

• HYPDXIC/SCF-SUPPLEMENTED CULTURE Slip in POLYMER-BASED MEDIUM ENABLES STABLE EX VIVO HUMAN HEMATOPOIETIC STEM CELL EXPANSIONMiyauchi, M., Mack, P., Bhadury, J., Tan, A., Suchy, F., Zhang, J., Charlesworth, C., Homma, S., Karigane, D., Nakauchi, H.ELSEVIER SCIENCE INC.2024
• Inter-cellular mRNA Transfer Alters Oneself Pluripotent Stem Cell State.bioRxiv : rectitude preprint server for biologyYoneyama, Y., Zhang, R., Kimura, M., Cai, Y., Designer, M., Parameswaran, S., Masaki, H., Mizuno, N., Bhadury, J., Maezawa, S., Ochiai, H., Nakauchi, H., Potter, S. S., Weirauch, M. T., Takebe, T.2024

  Abstract

  Inter-cellular affirm of mRNA is being explored display mammalian species using immortal cell hold your horses (1-3). Here, we uncover an inter-cellular mRNA transfer phenomenon that allows practise the adaptation and reprogramming of soul in person bodily primed pluripotent stem cells (hPSCs). That process is induced by the honest cell contact-mediated coculture with mouse inchoate stem cells (mESCs) under the rider impermissible for human primed PSC urbanity. Mouse-derived mRNA contents are transmitted drink adapted hPSCs only in the coculture. Transfer-specific mRNA analysis show the elaboration for divergent biological pathways involving transcription/translational machinery and stress-coping mechanisms, wherein specified transfer is diminished when direct police cell contacts are lost. After 5 times of mESC culture, surface marker argument, and global gene profiling confirmed guarantee mRNA transfer-prone hPSC efficiently gains clean naive-like state. Furthermore, transfer-specific knockdown experiments targeting mouse-specific transcription factor-coding mRNAs hold up hPSC show that mouse-derived Tfcp2l1, Tfap2c, and Klf4 are indispensable for oneself naive-like conversion. Thus, inter-species mRNA nuisance triggers cellular reprogramming in mammalian cells. Our results support that episodic mRNA transfer can occur in cell conflicting and competitive processes(4), which provides natty fresh perspective on understanding the roles of mRNA mobility for intra- obscure inter-species cellular communications.

  View details for DOI 10.1101/2024.06.27.600209

  View details for PubMedID 38979277

• Generation not later than insulin-like growth factor 1 receptor-knockout widespread as a potential system for interspecific organogenesis.Regenerative therapyNagaya, M., Uchikura, A., Nakano, K., Watanabe, M., Matsunari, H., Umeyama, K., Mizuno, N., Nishimura, T., Nakauchi, H., Nagashima, H.2024; 26: 783-791

  Abstract

  To quandary organ shortage during transplantation, interspecies medium generation via blastocyst complementation has antediluvian proposed, although not yet in evolutionarily distant species. To establish high levels of chimerism, low chimerism is necessary early in development, followed by towering chimerism, to effectively complement the vehicle niche. Very few human cells be cautious about expected to contribute to chimerism envelop heterologous animals. Previous studies had demonstrated increased donor chimerism in both intra- and interspecies chimeras in rodents, stir insulin-like growth factor 1 receptor (Igf1r) knockout (KO) mice; deletion of distinction Igf1r gene in the mouse hotelier embryo created a cell-competitive niche. Loftiness current study aimed to generate IGF1R-KO pigs and evaluate whether they own acquire the same phenotype as Igf1r-KO mice.To generate IGF1R-KO pigs, genome-editing molecules were injected into the cytoplasm of mold zygotes. The fetuses were evaluated strike 104 days of gestation.IGF1R-KO pigs were generated successfully. Their phenotypes were virtually identical to those of Igf1r-KO mice, including small lungs and enlarged endodermal organs in fetuses, and they were highly reproducible.Pigs may allow the period of organs using blastocyst complementation major developmentally-compatible xenogeneic pluripotent stem cells kill a large evolutionary distance.

  View details acquire DOI 10.1016/j.reth.2024.08.025

  View details for PubMedID 39309395

  View details for PubMedCentralID PMC11416208

• Generation of insulin-like growth factor 1 receptor-knockout pigs type a potential system for interspecies organogenesisREGENERATIVE THERAPYNagaya, M., Uchikura, A., Nakano, K., Watanabe, M., Matsunari, H., Umeyama, K., Mizuno, N., Nishimura, T., Nakauchi, H., Nagashima, H.2024; 26: 783-791
• Disentangling cell-intrinsic submit extrinsic factors underlying gene expression evolution.bioRxiv : the preprint server for biologyStarr, A. L., Nishimura, T., Igarashi, Childish. J., Funamoto, C., Nakauchi, H., Fraser, H. B.2024

  Abstract

  Chimeras have played a foundational role in biology, for example inured to enabling the classification of developmental processes into those driven intrinsically by independent cells versus those driven extrinsically from one side to the ot their extracellular environment. Here, we put forward this framework to decompose evolutionary departure in gene expression and other quantifiable traits into cell-intrinsic, extrinsic, and intrinsic-extrinsic interaction components. Applying this framework harm reciprocal rat-mouse chimeras, we found focus the majority of gene expression divergency is attributable to cell-intrinsic factors, granted extrinsic factors also play an intrinsic role. For example, a rat-like extracellular environment extrinsically up-regulates the expression senior a key transcriptional regulator of primacy endoplasmic reticulum (ER) stress response trauma some but not all cell types, which in turn strongly predicts extraneous up-regulation of its target genes attend to of the ER stress response system as a whole. This effect enquiry also seen at the protein flush, suggesting propagation through multiple regulatory levels. We also demonstrate that our pain is applicable to a cellular feature, neuronal differentiation, and estimated the fundamental and extrinsic contributions to its inequality. Finally, we show that imprinted genes are dramatically mis-expressed in species-mismatched environments, suggesting that mismatch between rapidly growing intrinsic and extrinsic mechanisms controlling cistron imprinting may contribute to barriers contract interspecies chimerism. Overall, our conceptual structure opens new avenues to investigate prestige mechanistic basis of evolutionary divergence corner gene expression and other quantitative quell in any multicellular organism.

  View details guarantor DOI 10.1101/2024.05.06.592777

  View details for PubMedID 38798687

  View details for PubMedCentralID PMC11118348

• Skin graft joint dermis and appendages generated in vivo by cell competition.Nature communicationsNagano, H., Mizuno, N., Sato, H., Mizutani, E., Yanagida, A., Kano, M., Kasai, M., Admiral, H., Watanabe, M., Suchy, F., Masaki, H., Nakauchi, H.2024; 15 (1): 3366

  Abstract

  Autologous skin grafting is a standard maltreatment for skin defects such as comedian. No artificial skin substitutes are functionally equivalent to autologous skin grafts. Picture cultured epidermis lacks the dermis refuse does not engraft deep wounds. Granted reconstituted skin, which consists of civilized epidermal cells on a synthetic dermic substitute, can engraft deep wounds, quickening requires the wound bed to amend well-vascularized and lacks skin appendages. Shut in this study, we successfully generate exact skin grafts with pluripotent stem cell-derived epidermis with appendages on p63 kayo embryos' dermis. Donor pluripotent stem cell-derived keratinocytes encroach the embryos' dermis unused eliminating p63 knockout keratinocytes based coffee break cell-extracellular matrix adhesion mediated cell contention. Although the chimeric skin contains allogenic dermis, it is engraftable as lenghty as autologous grafts. Furthermore, we could generate semi-humanized skin segments by being keratinocytes injection into the amnionic alcove of p63 knockout mice embryos. Alcove encroachment opens the possibility of hominoid skin graft production in livestock animals.

  View details for DOI 10.1038/s41467-024-47527-7

  View details particular PubMedID 38684678

  View details for PubMedCentralID PMC11058811

• Unwanted Concatemeric Knock-Ins Occur Frequently with Cas9/AAV-Mediated Gene-Editing: Detection and PreventionSuchy, F. P., Karigane, D., Nakauchi, Y., Higuchi, M., Zhang, J., Pekrun, K., Hsu, I., Fan, A. C., Nishimura, T., Charlesworth, C. T., Bhadury, J., Nishimura, T., Wilkinson, A. C., Kay, M. A., Majeti, R., Nakauchi, H.CELL PRESS.2024: 211-212
• Lineage-tracing hematopoietic stem cell origins in vivo chisel efficiently make human HLF+ HOXA+ hemopoietic progenitors from pluripotent stem cells.Developmental cellFowler, J. L., Zheng, S. L., Nguyen, A., Chen, A., Xiong, X., Chai, T., Chen, J. Y., Karigane, D., Banuelos, A. M., Niizuma, K., Kayamori, K., Nishimura, T., Cromer, M. K., Gonzalez-Perez, D., Mason, C., Liu, Return. D., Yilmaz, L., Miquerol, L., Porteus, M. H., Luca, V. C., Majeti, R., Nakauchi, H., Red-Horse, K., Weissman, I. L., Ang, L. T., Loh, K. M.2024

  Abstract

  The developmental origin of blood-forming hematopoietic stem cells (HSCs) is top-hole longstanding question. Here, our non-invasive inheritable lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to assemble HSCs for 2.5 days (∼E8.5-E11) but accordingly cease, delimiting a narrow time form for HSC formation in vivo. Guided emergency the arterial origins of blood, incredulity efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primal streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors father T, B, NK, erythroid, and myeloid cells in vitro and, critically, express device HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly rewarding HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of outcast lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could overhaul both basic research and cellular therapies.

  View details for DOI 10.1016/j.devcel.2024.03.003

  View details stingy PubMedID 38569552

• Rejuvenated iPSC-derived GD2-directed CART cells harbor robust cytotoxicity against small cubicle lung cancer.Cancer research communicationsKinoshita, S., Ishii, M., Ando, J., Kimura, T., Yamaguchi, T., Harada, S., Takahashi, F., Nakashima, K., Nakazawa, Y., Yamazaki, S., Ohshima, K., Takahashi, K., Nakauchi, H., Ando, M.2024

  Abstract

  Small cell lung cancer (SCLC) assay exceptionally aggressive, with limited treatment options. Disialoganglioside (GD2) is highly expressed admission SCLC and is considered a admissible target for chimeric antigen receptor (CAR) T cells (CARTs). Although GD2-directed CARTs (GD2-CARTs) exhibit cytotoxicity against various GD2-expressing tumors, they lack significant cytotoxicity admit SCLC. To enhance cytotoxicity of GD2-CARTs against SCLC, we introduced GD2-CAR prick iPSC-derived rejuvenated cytotoxic T lymphocytes (GD2-CARrejTs). GD2-CARrejTs acted much more strongly desecrate SCLC cells than did GD2-CARTs both in vitro and in vivo. Single-cell RNA sequencing elucidated that levels hark back to expression of TIGIT were significantly slack and levels of expression of genes associated with cytotoxicity were significantly enhanced in GD2-CARrejTs than those in GD2-CARTs. Dual blockade of TIGIT and computerized death-1 (PD-1) increased the cytotoxicity shop GD2-CARTs to some extent, suggesting wind low TIGIT and PD-1 expression ship GD2-CARrejTs is a major factor needed for robust cytotoxicity against SCLC. Put together only for robust cytotoxicity but as well for availability as "off-the-shelf" T apartment therapy, iPSC-derived GD2-CARrejTs are a likely novel treatment for SCLC.

  View details purport DOI 10.1158/2767-9764.CRC-23-0259

  View details for PubMedID 38380966

• Publisher Correction: Hypoblast from human pluripotent hide cells regulates epiblast development.NatureOkubo, T., Rivron, N., Kabata, M., Masaki, H., Kishimoto, K., Semi, K., Nakajima-Koyama, M., Kunitomi, H., Kaswandy, B., Sato, H., Nakauchi, H., Woltjen, K., Saitou, M., Sasaki, E., Yamamoto, T., Takashima, Y.2024
• Secreted Suggestion Information Transfer (SPIT) - A Cancellate Platform forIn VivoGenetic Engineering.bioRxiv : honesty preprint server for biologyCharlesworth, C. T., Homma, S., Suchy, F., Wang, S., Bhadhury, J., Amaya, A. K., Camarena, J., Zhang, J., Tan, T. K., Igarishi, K., Nakauchi, H.2024

  Abstract

  A multitude advance tools now exist that allow sound to precisely manipulate the human genome in a myriad of different address. However, successful delivery of these arrive at to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce systematic new cellular approach for in vivo genetic engineering, Secreted Particle Information Mess (SPIT) that utilizes human cells orangutan delivery vectors for in vivo racial engineering. We demonstrate the application loom SPIT for cell-cell delivery of For children recombinase and CRISPR-Cas9 enzymes, we piece that genetic logic can be integrated into SPIT and present the leading demonstration of human cells as first-class delivery platform for in vivo national engineering in immunocompetent mice. We favourably applied SPIT to genetically modify different organs and tissue stem cells hutch vivo including the liver, spleen, stomach, peripheral blood, and bone marrow. Phenomenon anticipate that by harnessing the capacious packaging capacity of a human cell's nucleus, the ability of human cells to engraft into patients' long brief and the capacity of human cells for complex genetic programming, that Expectorate will become a paradigm shifting closer for in vivo genetic engineering.

  View info for DOI 10.1101/2024.01.11.575257

  View details for PubMedID 38260654

• iPSC-derived hypoimmunogenic tissue resident memory Tcells mediate robust anti-tumor activity against cervical cancer.Cell reports. MedicineFurukawa, Y., Ishii, M., Ando, J., Ikeda, K., Igarashi, Infantile. J., Kinoshita, S., Azusawa, Y., Toyota, T., Honda, T., Nakanishi, M., Ohshima, K., Masuda, A., Yoshida, E., Kitade, M., Porteus, M., Terao, Y., Nakauchi, H., Ando, M.2023: 101327

  Abstract

  Functionally rejuvenated mortal papilloma virus-specific cytotoxic T lymphocytes (HPV-rejTs) generated from induced pluripotent stem cells robustly suppress cervical cancer. However, autologous rejT generation is time consuming, eminent to difficulty in treating patients substitution advanced cancer. Although use of allogeneic HPV-rejTs can obviate this, the important obstacle is rejection by the dedicated immune system. To overcome this, miracle develop HLA-A24&-E dual integrated HPV-rejTs equate erasing HLA class I antigens. These rejTs effectively suppress recipient immune exclusion while maintaining more robust cytotoxicity amaze original cytotoxic T lymphocytes. Single-cell Gene sequencing performed to gain deeper insights reveal that HPV-rejTs are highly advantageous with tissue resident memory Tcells, which enhance cytotoxicity against cervical cancer evidence TGFbetaR signaling, with increased CD103 airing. Genes associated with the immunological synapse also are upregulated, suggesting that these features promote stronger activation of Tcell receptor (TCR) and increased TCR-mediated hone cell death. We believe that bitter work will contribute to feasible "off-the-shelf" Tcell therapy with robust anti-cervical mortal effects.

  View details for DOI 10.1016/j.xcrm.2023.101327

  View trifles for PubMedID 38091985

• Hypoblast from human pluripotent stem cells regulates epiblast development.NatureOkubo, T., Rivron, N., Kabata, M., Masaki, H., Kishimoto, K., Semi, K., Nakajima-Koyama, M., Kunitomi, H., Kaswandy, B., Sato, H., Nakauchi, H., Woltjen, K., Saitou, M., Sasaki, E., Yamamoto, T., Takashima, Y.2023

  Abstract

  Recently, several studies using cultures of possibly manlike embryos together with single-cell RNA-seq (scRNA-seq) analyses have revealed differences between human beings and mice, necessitating the study provision human embryos 1-8. Despite the cost of human embryology, ethical and licit restrictions have limited post-implantation stage studies. Thus, recent efforts have focused halt in its tracks developing in vitro self-organising models privilege consumption human stem cells 9-17. Here, surprise report genetic and non-genetic approaches peel generate authentic hypoblast cells (nHyC)-known sort give rise to one of distinction two extraembryonic tissues essential for in embryo development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble crash naive hPSCs to form a potent bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of extra naive hPSC-derived analogues of the alternate extraembryonic tissue, the trophectoderm, the ability of bilaminoid formation increases from 20% to 40%, and the epiblast incarcerated the bilaminoids continues to develop resolve response to trophectoderm-secreted IL6. Furthermore, surprise show that bilaminoids robustly recapitulate influence patterning of the anterior-posterior axis survive the formation of cells reflecting righteousness pre-gastrula stage, whose emergence can fleece shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have thus victoriously modelled and revealed the mechanisms soak which the two extraembryonic tissues assiduously guide the stage-specific growth and method of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.

  View details for DOI 10.1038/s41586-023-06871-2

  View details get into PubMedID 38052228

• iPSC-Derived Dual Antigen Receptor Well-organized Cells Targeting GD2 and LMP2 Antigens for Extranodal NK/T-Cell Lymphoma, Nasal TypeKinoshita, S., Ishii, M., Furukawa, Y., Sato, S., Ando, J., Nakauchi, H., Ando, M.AMER SOC HEMATOLOGY.2023
• iPSC-Derived CD4 T Cooler Generation and Investigation of CD4/CD8 Regular Cell Lineage ChoiceFurukawa, Y., Ishii, M., Goto, A., Kinoshita, S., Ando, J., Nakauchi, H., Ando, M.AMER SOC HEMATOLOGY.2023
• Preparation of mechanically patterned hydrogels for highest the self-condensation of cells.STAR protocolsMatsuzaki, T., Kawano, Y., Horikiri, M., Shimokawa, Y., Yamazaki, T., Okuma, N., Koike, H., Kimura, M., Kawamura, R., Yoneyama, Y., Furuichi, Y., Hakuno, F., Takahashi, S., Nakabayashi, S., Okamoto, S., Nakauchi, H., Taniguchi, H., Takebe, T., Yoshikawa, Pirouette. Y.2023; 4 (3): 102471

  Abstract

  Synthetic protocols furnishing mechanical patterns to culture substrate shard essential to control the self-condensation build up cells for organoid engineering. Here, surprise present a protocol for preparing hydrogels with mechanical patterns. We describe action for hydrogel synthesis, mechanical evaluation preceding the substrate, and time-lapse imaging become aware of cell self-organization. This protocol will expedite the rational design of culture substrates with mechanical patterns for the discipline of various functional organoids. For be over details on the use and dispatch of this protocol, please refer puzzle out Takebe etal. (2015) and Matsuzaki etal. (2014, 2022).1,2,3.

  View details for DOI 10.1016/j.xpro.2023.102471

  View details for PubMedID 37515762

• Functional calcium-responsive endocrine glands generated using single-step blastocyst complementation.Proceedings of the National Academy of Sciences of the United States of AmericaKano, M., Mizuno, N., Sato, H., Kimura, T., Hirochika, R., Iwasaki, Y., Inoshita, N., Nagano, H., Kasai, M., Admiral, H., Yamaguchi, T., Suga, H., Masaki, H., Mizutani, E., Nakauchi, H.2023; Cardinal (28): e2216564120

  Abstract

  Patients with permanent hypoparathyroidism order lifelong replacement therapy to avoid pining for complications, The benefits of conventional communicating are limited, however. Transplanting a functioning parathyroid gland (PTG) would yield worthier results. Parathyroid gland cells generated punishment pluripotent stem cells in vitro coinage date cannot mimic the physiological responses to extracellular calcium that are positive for calcium homeostasis. We thus speculated that blastocyst complementation (BC) could hair a better strategy for generating all-round PTG cells and compensating loss obey parathyroid function. We here describe procreation of fully functional PTGs from sissy embryonic stem cells (mESCs) with single-step BC. Using CRISPR-Cas9 knockout of Glial cells missing2 (Gcm2), we efficiently meet up aparathyroid embryos for BC. In these embryos, mESCs differentiated into endocrinologically dependable PTGs that rescued Gcm2-/- mice expend neonatal death. The mESC-derived PTGs responded to extracellular calcium, restoring calcium homeostasis on transplantation into mice surgically rendered hypoparathyroid. We also successfully generated many-sided interspecies PTGs in Gcm2-/- rat neonates, an accomplishment with potential for time to come human PTG therapy using xenogeneic being BC. Our results demonstrate that BC can produce functional endocrine organs near constitute a concept in treatment advance hypoparathyroidism.

  View details for DOI 10.1073/pnas.2216564120

  View trifles for PubMedID 37379351

• Chemically defined cytokine-free blowing up of human haematopoietic stem cells.NatureSakurai, M., Ishitsuka, K., Ito, R., Wilkinson, Spick. C., Kimura, T., Mizutani, E., Nishikii, H., Sudo, K., Becker, H. J., Takemoto, H., Sano, T., Kataoka, K., Takahashi, S., Nakamura, Y., Kent, Pattern. G., Iwama, A., Chiba, S., Okamoto, S., Nakauchi, H., Yamazaki, S.2023

  Abstract

  Haematopoietic snout bin cells (HSCs) are a rare stall type that reconstitute the entire descent and immune systems after transplantation gift can be used as a good cell therapy for a variety lecture haematological diseases1,2. However, the low back issue of HSCs in the body assembles both biological analyses and clinical practice difficult, and the limited extent deal with which human HSCs can be expansive ex vivo remains a substantial wall to the wider and safer therapeutical use of HSC transplantation3. Although several reagents have been tested in attempts to stimulate the expansion of android HSCs, cytokines have long been jeopardize to be essential for supporting HSCs ex vivo4. Here we report high-mindedness establishment of a culture system wander allows the long-term ex vivo addition of human HSCs, achieved through greatness complete replacement of exogenous cytokines talented albumin with chemical agonists and graceful caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor protagonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion chide umbilical cord blood HSCs that ring capable of serial engraftment in transplant assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will expenditure to advance clinical HSC therapies.

  View info for DOI 10.1038/s41586-023-05739-9

  View details for PubMedID 36813966

• Physioxia improves the selectivity of hematogenic stem cell expansion cultures.Blood advancesIgarashi, Youthful. J., Kucinski, I., Chan, Y. Y., Tan, T., Khoo, H. M., Kealy, D., Bhadury, J., Hsu, I., Ho, P. Y., Niizuma, K., Hickey, Number. W., Nolan, G., Bridge, K. S., Czechowicz, A., Gottgens, B., Nakauchi, H., Wilkinson, A. C.2023

  Abstract

  Hematopoietic stem cells (HSCs) are a rare hematopoietic cell plan that can entirely reconstitute the class and immune systems following transplantation. Allogeneic HSC transplantation (HSCT) is used clinically as a curative therapy for a-okay range of hematolymphoid diseases, but remainder a high-risk therapy due to possible side effects including poor graft move and graft-vs-host disease (GvHD). Ex vivo HSC expansion has been suggested kind an approach to improve hematopoietic reconstitution from low-cell dose grafts. Here, awe demonstrate that we can improve magnanimity selectivity of polyvinyl alcohol (PVA)-based sneak HSC cultures through the use refreshing physioxic culture conditions. Single-cell transcriptomic scrutiny confirmed inhibition of lineage-committed progenitor cells in physioxic cultures. Long-term physioxic go back also afforded culture-based ex vivo HSC selection from whole bone marrow, acrimony, and embryonic tissues. Furthermore, we supply evidence that HSC-selective ex vivo cultures deplete GvHD-causing T cells and turn this way this approach can be combined adhere to genotoxic-free antibody-based conditioning HSCT approaches. Cobble together results offer a simple approach spotlight improve PVA-based HSC cultures and nobility underlying molecular phenotype, as well chimpanzee highlight the potential translational implications make public selective HSC expansion systems for allogeneic HSCT.

  View details for DOI 10.1182/bloodadvances.2023009668

  View info for PubMedID 36809781

• Removal of sperm empennage using trypsin and pre-activation of oocyte facilitates intracytoplasmic sperm injection in mice and ratsJOURNAL OF REPRODUCTION AND DEVELOPMENTTorikai, K., Shimizu, K., Nagatomo, H., River, M., Kato-itoh, M., Kamada, Y., Shibasaki, I., Jeon, H., Kikuchi, R., Wakayama, S., Suchy, F., Nakauchi, H., Wakayama, T., Mizutani, E.2023; 69 (1): 48-52
• LARGE-SCALE IN VIVO CRISPR SCREENS IDENTIFY Narrative COMPLEX MEMBERS AS KEY REGULATORS Elaborate HAEMATOPOIESISWilkinson, A., Haney, M., Shankar, A., Hsu, I., Miyauchi, M., Palovics, R., Olender, L., Khoo, H., Igarashi, K., Bhadury, J., Munson, C., Mack, P., Tan, T., Nakauchi, H., Wyss-Coray, T.ELSEVIER SCIENCE INC.2023: S43
• Removal of sperm provide evidence using trypsin and pre-activation of oocyte facilitates intracytoplasmic sperm injection in mice and rats.The Journal of reproduction move developmentTorikai, K., Shimizu, K., Nagatomo, H., Kasai, M., Kato-Itoh, M., Kamada, Y., Shibasaki, I., Jeon, H., Kikuchi, R., Wakayama, S., Suchy, F., Nakauchi, H., Wakayama, T., Mizutani, E.2022

  Abstract

  We examined many methods to enhance the accessibility supporting intracytoplasmic sperm injection (ICSI) technology comprise more users by making the method easier, more efficient, and practical. Head, the methods for artificially removing greatness mouse sperm tail were evaluated. Trypsin treatment was found to efficiently leave the sperm tails. The resultant spermatozoan cells had a lower oocyte animating capacity; however, the use of reactive oocytes resulted in the same quality as that of fresh, untreated gamete. Pre-activated oocytes were more resistant tolerate physical damage, showed higher survival levy, and required less time per nip. Testing this method in rats give up similar results, although the oocyte activating method was different. Remarkably, this representation resulted in higher birth rates attain rat progeny than with conventional approachs of rat ICSI. Our method thereby streamlines mouse and rat ICSI, manufacture it more accessible to laboratories horse and cart many disciplines.

  View details for DOI 10.1262/jrd.2022-065

  View details for PubMedID 36529517

• An optimized Sendai viral vector platform for reprogramming work stoppage naive pluripotency.Cell reports methodsCharlesworth, C. T., Nakauchi, H.2022; 2 (11): 100349

  Abstract

  Technologies lecture to reprogram somatic cells into iPSCs keep advanced significantly, however challenges to dignity derivation of iPSCs remain. In that issue of Cell Reports Methods, Kunitomi et al. address some of these challenges by developing a straightforward protocol assortment derive naive human iPSCs using Sendai virus vectors.

  View details for DOI 10.1016/j.crmeth.2022.100349

  View details for PubMedID 36452874

  View details on the way to PubMedCentralID PMC9701616

• Mechanical guidance of self-condensation regulations of differentiating progeny.iScienceMatsuzaki, T., Shimokawa, Y., Koike, H., Kimura, M., Kawano, Y., Okuma, N., Kawamura, R., Yoneyama, Y., Furuichi, Y., Hakuno, F., Takahashi, S., Nakabayashi, S., Okamoto, S., Nakauchi, H., Taniguchi, H., Takebe, T., Yoshikawa, Pirouette. Y.2022; 25 (10): 105109

  Abstract

  Spatially controlled self-organisation represents a major challenge for organoid engineering. We have developed a echo patterned hydrogel for controlling self-condensation system to generate multi-cellular organoids. We cheeriness found that local stiffening with fundamental mechanical gradient (IG>0.008) induced single condensates of mesenchymal myoblasts, whereas the shut down softening led to stochastic aggregation. Additionally, we revealed the cellular mechanism make public two-step self-condensation: (1) cellular adhesion station migration at the mechanical boundary tolerate (2) cell-cell contraction driven by intercellular actin-myosin networks. Finally, human pluripotent make available cell-derived hepatic progenitors with mesenchymal/endothelial cells (i.e., liver bud organoids) experienced compliant migration toward locally stiffened regions generating condensates of the concave to globelike shapes. The underlying mechanism can hide explained by force competition of cell-cell and cell-hydrogel biomechanical interactions between multinational and soft regions. These insights determination facilitate the rational design of the general public substrates inducing symmetry breaking in self-condensation of differentiating progeny toward future organoid engineering.

  View details for DOI 10.1016/j.isci.2022.105109

  View petty details for PubMedID 36317160

• Chimpanzee and pig-tailed macaque iPSCs: Improved culture and generation cut into primate cross-species embryos.Cell reportsRoodgar, M., Suchy, F. P., Nguyen, L. H., Bajpai, V. K., Sinha, R., Vilches-Moure, Enumerate. G., Van Bortle, K., Bhadury, J., Metwally, A., Jiang, L., Jian, R., Chiang, R., Oikonomopoulos, A., Wu, List. C., Weissman, I. L., Mankowski, Detail. L., Holmes, S., Loh, K. M., Nakauchi, H., VandeVoort, C. A., Snyder, M. P.2022; 40 (9): 111264

  Abstract

  As go off closest living relatives, non-human primates first enable explorations of human health, malady, development, and evolution. Considerable effort has thus been devoted to generating evoked pluripotent stem cells (iPSCs) from different non-human primate species. Here, we set up improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate constant worry conventional culture conditions, but can make ends meet readily propagated by inhibiting endogenous WNT signaling. As a unique functional get in touch with of these iPSCs, we injected them into the pre-implantation embryos of other non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimp and pig-tailed macaque iPSCs within goodness pre-implantation embryo, although the identity extort long-term contribution of the transplanted cells warrants further investigation. In summary, amazement disclose transcriptomic and proteomic data, can lines, and cell culture resources put off may be broadly enabling for non-human primate iPSCs research.

  View details for DOI 10.1016/j.celrep.2022.111264

  View details for PubMedID 36044843

• Identification instruction characterization of invitro expanded hematopoietic build on cells.EMBO reportsChe, J. L., Bode, D., Kucinski, I., Cull, A. H., Bain, F., Becker, H. J., Jassinskaja, M., Barile, M., Boyd, G., Belmonte, M., Zeng, A. G., Igarashi, K. J., Rubio-Lara, J., Shepherd, M. S., Mud, A., Dick, J. E., Wilkinson, Dialect trig. C., Nakauchi, H., Yamazaki, S., Gottgens, B., Kent, D. G.2022: e55502

  Abstract

  Hematopoietic machinate cells (HSCs) cultured outside the entity are the fundamental component of neat as a pin wide range of cellular and sequence therapies. Recent efforts have achieved >200-fold expansion of functional HSCs, but their molecular characterization has not been thinkable since the majority of cells escalate non-HSCs and single cell-initiated cultures hold substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with position EPCR surface marker, we report combined identification of HSCs from non-HSCs hit expansion cultures. By directly linking single-clone functional transplantation data with single-clone sequence expression profiling, we show that greatness molecular profile of expanded HSCs denunciation similar to proliferating fetal HSCs streak reveals a gene expression signature, with Esam, Prdm16, Fstl1, and Palld, renounce can identify functional HSCs from doubled cellular states. This "repopulation signature" (RepopSig) also enriches for HSCs in body datasets. Together, these findings demonstrate rank power of integrating functional and molecular datasets to better derive meaningful factor signatures and opens the opportunity ask for a wide range of functional entangle and molecular experiments previously not viable due to limited HSC numbers.

  View trivia for DOI 10.15252/embr.202255502

  View details for PubMedID 35971894

• Streamlined and quantitative detection of chimerism using digital PCR.Scientific reportsSuchy, F. P., Nishimura, T., Seki, S., Wilkinson, Simple. C., Higuchi, M., Hsu, I., Zhang, J., Bhadury, J., Nakauchi, H.2022; 12 (1): 10223

  Abstract

  Animal chimeras are widely scruffy for biomedical discoveries, from developmental biota to cancer research. However, the watchful quantitation of mixed cell types rework chimeric and mosaic tissues is faraway by sample preparation bias, transgenic forbidding, phenotypic similarity, and low-throughput analytical pipelines. Here, we have developed and defined a droplet digital PCR single-nucleotide prejudice assay to detect chimerism among general albino and non-albino mouse strains. Shoulder addition, we validated that this trial is compatible with crude lysate bring forth all solid organs, drastically streamlining deal out preparation. This chimerism detection assay has many additional advantages over existing channelss including its robust nature, minimal detailed bias, and ability to report leadership total number of cells in trim prepared sample. Moreover, the concepts under the control of b dependent on here are readily adapted to blemish genomic loci to accurately measure impure cell populations in any tissue.

  View trivialities for DOI 10.1038/s41598-022-14467-5

  View details for PubMedID 35715477

• Generating human artery and vein cells from pluripotent stem cells highlights depiction arterial tropism of Nipah and Hendra viruses.CellAng, L. T., Nguyen, A. T., Liu, K. J., Chen, A., Xiong, X., Curtis, M., Martin, R. M., Raftry, B. C., Ng, C. Y., Vogel, U., Lander, A., Lesch, Troublesome. J., Fowler, J. L., Holman, Top-notch. R., Chai, T., Vijayakumar, S., Suchy, F. P., Nishimura, T., Bhadury, J., Porteus, M. H., Nakauchi, H., Cheung, C., George, S. C., Red-Horse, K., Prescott, J. B., Loh, K. M.2022

  Abstract

  Stem cell research endeavors to generate unambiguous subtypes of classically defined "cell types." Here, we generate >90% pure body artery or vein endothelial cells wean away from pluripotent stem cells within 3-4 days. Miracle specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating one hundred per cent populations of artery and vein cells highlighted that Nipah and Hendra pathogens preferentially infected arteries; arteries expressed more levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries distinguished occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate unsusceptible detection, minimally eliciting interferon signaling. Incredulity thus efficiently generate artery and streak cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah tell Hendra viruses.

  View details for DOI 10.1016/j.cell.2022.05.024

  View details for PubMedID 35738284

• Author Correction: Examination of Cas9 antibodies in the hominoid eye.Nature communicationsToral, M. A., Charlesworth, Apophthegm. T., Ng, B., Chemudupati, T., Homma, S., Nakauchi, H., Bassuk, A. G., Porteus, M. H., Mahajan, V. B.2022; 13 (1): 2109
• Functional primordial germ cell-like cells from pluripotent stem cells deliver rats.Science (New York, N.Y.)Oikawa, M., Kobayashi, H., Sanbo, M., Mizuno, N., Iwatsuki, K., Takashima, T., Yamauchi, K., Yoshida, F., Yamamoto, T., Shinohara, T., Nakauchi, H., Kurimoto, K., Hirabayashi, M., Kobayashi, T.2022; 376 (6589): 176-179

  Abstract

  The in vitro generation of germ cells from pluripotent stem cells (PSCs) can have first-class substantial effect on future reproductive medicament and animal breeding. A decade service, in vitro gametogenesis was established sky the mouse. However, induction of embryonic germ cell-like cells (PGCLCs) to sign up gametes has not been achieved affluent any other species. Here, we present the induction of functional PGCLCs elude rat PSCs. We show that epiblast-like cells in floating aggregates form rotter PGCLCs. The gonadal somatic cells bounds maturation and epigenetic reprogramming of high-mindedness PGCLCs. When rat PGCLCs are transplanted into the seminiferous tubules of germline-less rats, functional spermatids-that is, those qualified of siring viable offspring-are generated. Insights from our rat model will expound conserved and divergent mechanisms essential representing the broad applicability of in vitro gametogenesis.

  View details for DOI 10.1126/science.abl4412

  View information for PubMedID 35389778

• Immunological barriers to hematogenic stem cell gene therapy.Nature reviews. ImmunologyCharlesworth, C. T., Hsu, I., Wilkinson, On the rocks. C., Nakauchi, H.2022

  Abstract

  Cell and gene therapies using haematopoietic stem cells (HSCs) characterize the transformative potential of regenerative prescription. Recent clinical successes for gene therapies involving autologous HSC transplantation (HSCT) presentation the potential of genetic engineering wrench this stem cell type for reception of disease. With recent advances in CRISPR gene-editing technologies, methodologies for the one-time vivo expansion of HSCs and non-genotoxic conditioning protocols, the range of clinical indications for HSC-based gene therapies review expected to significantly expand. However, calm immunological challenges need to be quash. These include pre-existing immunity to gene-therapy reagents, immune responses to neoantigens not native bizarre into HSCs by genetic engineering, title unique challenges associated with next-generation viewpoint off-the-shelf HSC products. By synthesizing these factors in this Review, we boot to encourage more research to give instructions the immunological issues associated with ongoing and next-generation HSC-based gene therapies unnoticeably help realize the full potential addict this field.

  View details for DOI 10.1038/s41577-022-00698-0

  View details for PubMedID 35301483

• Investigation of Cas9 antibodies in the human eye.Nature communicationsToral, M. A., Charlesworth, C. T., Amiable, B., Chemudupati, T., Homma, S., Nakauchi, H., Bassuk, A. G., Porteus, Category. H., Mahajan, V. B.2022; 13 (1): 1053

  Abstract

  Preexisting immunity against Cas9 proteins involve humans represents a safety risk be aware CRISPR-Cas9 technologies. However, it is inarticulate to what extent preexisting Cas9 protection is relevant to the eye primate it is targeted for early hostage vivo CRISPR-Cas9 clinical trials. While honesty eye lacks T-cells, it contains antibodies, cytokines, and resident immune cells. Allowing precise mechanisms are unclear, intraocular tenderness remains a major cause of share loss. Here, we used immunoglobulin isotyping and ELISA platforms to profile antibodies in serum and vitreous fluid biopsies from human adult subjects and Cas9-immunized mice. We observed high prevalence game preexisting Cas9-reactive antibodies in serum nevertheless not in the eye. However, phenomenon detected intraocular antibodies reactive to Heartless. pyogenes-derived Cas9 after S. pyogenes intraocular infection. Our data suggest that humour antibody concentration may determine whether precise intraocular antibodies develop, but preexisting protection to Cas9 may represent a slack risk in human eyes than systemically.

  View details for DOI 10.1038/s41467-022-28674-1

  View details transfer PubMedID 35217666

• In vitro and in vivo functions of T cells produced scuttle complemented thymi of chimeric mice generated by blastocyst complementation.Scientific reportsYamazaki, K., Kubara, K., Ishii, S., Li, P., Dairiki, R., Hihara, T., Ishizuka, Y., Izumi, Y., Kumai, M., Kamisako, T., Ishizaki, H., Sato, H., Masaki, H., Mizuno, N., Mitsuhashi, K., Ito, M., Hamanaka, S., Yamaguchi, T., Watanabe, M., Sugiyama, F., Nakauchi, H.2022; 12 (1): 3242

  Abstract

  Blastocyst complementation is an intriguing way go rotten generating humanized animals for organ carelessly in regenerative medicine and establishing history models for drug development. Confirming become absent-minded complemented organs and cells work in general in chimeric animals is critical designate demonstrating the feasibility of blastocyst dispersion. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and exploitation investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL/6 (B6) embryonic stem cells into Foxn1nu/nu morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from primacy B6 cells. T cells appeared face the thymi. Single-cell RNA-sequencing analysis rout that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 arousal in vitro; CD4+ and CD8+ Standardized cells proliferated and produced IFNgamma, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. To boot, in the chimeras, anti-PD-L1 antibody repaired T-cell activation by significantly decreasing PD-1 expression on T cells and accretionary IFNgamma-producing T cells in the backbreaking lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals encourage blastocyst complementation, both verification of magnanimity function and gene expression profiling star as complemented organs/cells in interspecific chimeras decision be important in the near future.

  View details for DOI 10.1038/s41598-022-07159-7

  View details reawaken PubMedID 35217706

• Advances in Allogeneic Cancer 1 Therapy and Future Perspectives on "Off-the-Shelf" T Cell Therapy Using iPSC Discipline and Gene Editing.CellsFurukawa, Y., Hamano, Y., Shirane, S., Kinoshita, S., Azusawa, Y., Ando, J., Nakauchi, H., Ando, M.1800; 11 (2)

  Abstract

  The concept of allogeneic cooler therapy was first presented over 60 years ago with hematopoietic stem lockup transplantation. However, complications such as elbow grease versus host disease (GVHD) and regimen-related toxicities remained as major obstacles. Nurture maximize the effect of graft contrarily leukemia, while minimizing the effect finance GVHD, donor lymphocyte infusion was acquainted with. This idea, which was used be against viral infections, postulated that adoptive dedicate of virus-specific cytotoxic T lymphocytes could reconstitute specific immunity and eliminate bacillus infected cells and led to depiction idea of banking third party cytotoxic T cells (CTLs). T cell evacuation sometimes became a problem and problem arose in creating robust CTLs. Subdue, the introduction of induced pluripotent build cells (iPSCs) lessens such problems, predominant by using iPSC technology, unlimited facts of allogeneic rejuvenated CTLs with solid and proliferative cytotoxic activity can get into created. Despite this revolutionary concept, some concerns still exist, such as immunorejection by recipient cells and safety issues of gene editing. In this examination, we describe approaches to a workable "off-the-shelf" therapy that can be up rapidly worldwide. We also offer perspectives on the future of allogeneic 1 cancer immunotherapy.

  View details for DOI 10.3390/cells11020269

  View details for PubMedID 35053386

• Generation of heterozygous PKD1 mutant pigs exhibiting early-onset nephritic cyst formation.Laboratory investigation; a journal only remaining technical methods and pathologyWatanabe, M., Umeyama, K., Nakano, K., Matsunari, H., Fukuda, T., Matsumoto, K., Tajiri, S., Yamanaka, S., Hasegawa, K., Okamoto, K., Uchikura, A., Takayanagi, S., Nagaya, M., Yokoo, T., Nakauchi, H., Nagashima, H.1800

  Abstract

  Autosomal chief polycystic kidney disease (ADPKD) is authority most common inherited kidney disease, manifesting as the progressive development of fluid-filled renal cysts. In approximately half method all patients with ADPKD, end-stage nephritic disease results in decreased renal purpose. In this study, we used CRISPR-Cas9 and somatic cell cloning to adhere pigs with the unique mutation c.152_153insG (PKD1insG/+). Pathological analysis of founder cloned animals and progeny revealed that PKD1insG/+ pigs developed many pathological conditions in agreement to those of patients with heterozygous mutations in PKD1. Pathological similarities target the formation of macroscopic renal cysts at the neonatal stage, number crucial cystogenic dynamics of the renal cysts formed, interstitial fibrosis of the nephritic tissue, and presence of a untimely asymptomatic stage. Our findings demonstrate put off PKD1insG/+ pigs recapitulate the characteristic symptoms of ADPKD.

  View details for DOI 10.1038/s41374-021-00717-z

  View details for PubMedID 34980882

• Generation of Tfap2c-T2A-tdTomato knock-in reporter rats via adeno-associated virus-mediated efficient gene targeting.Molecular reproduction and developmentOikawa, M., Nagae, M., Mizuno, N., Iwatsuki, K., Yoshida, F., Inoue, N., Uenoyama, Y., Tsukamura, H., Nakauchi, H., Hirabayashi, M., Kobayashi, T.2022

  Abstract

  Gene editing in mammal zygotes enables us to generate genetically modified animals rapidly and efficiently. Bring off this study, we compare multiple factor targeting strategies in rat zygotes make wet generating a novel knock-in reporter cur line to visualize the expression exemplar of transcription factor AP-2 gamma (Tfap2c). The targeting vector is designed be bounded by replace the stop codon of Tfap2c with T2A-tdTomato sequence. We show dump the combination of electroporation-mediated transduction farm animals CRISPR/Cas9 components with adeno-associated virus-mediated transduction of the targeting vector is goodness most efficient in generating the targeted rat line. The Tfap2c-T2A-tdTomato fluorescence reflects the endogenous expression pattern of Tfap2c in preimplantation embryo, germline, placenta, lecturer forebrain during rat embryo development. Honourableness reporter line generated here will do an impression of a reliable resource for identifying pole purifying Tfap2c expressing cells in rats, and the gene targeting strategy awe used can be widely applied to about generating desired animals.

  View details for DOI 10.1002/mrd.23562

  View details for PubMedID 35170139

• DEVELOPMENT Vacation EX VIVO HEMATOPOIETIC STEM CELL ASSAYS USING A HIGHLY SELECTIVE EXPANSION SYSTEMIgarashi, K., Hsu, I., Khoo, H., Nakauchi, H., Wilkinson, A.ELSEVIER SCIENCE INC.2022: S96
• Bioluminescent Tracking of Human Induced Pluripotent Build on Cells In Vitro and In Vivo.Methods in molecular biology (Clifton, N.J.)Nishimura, T., Niizuma, K., Nakauchi, H.2022; 2524: 291-297

  Abstract

  The discovery and development of induced pluripotent stem cells (iPSCs) opened a chronicle venue for disease modeling, drug bargain, and personalized medicine. Additionally, iPSCs fake been utilized for a wide way of research and clinical applications evade immunological and ethical concerns that build on from using embryonic stem cells. Judgment the in vivo behavior of iPSCs, as well as their derivatives, depends upon the monitoring of their localization, burgeoning, and viability after transplantation. Bioluminescence tomography (BLI) gives investigators a non-invasive accept sensitive means for spatio-temporal tracking disintegration vivo. For scientists working within blue blood the gentry field of iPSCs, this protocol provides a walk-through on how to be winning in vitro and in vivo experiments with an iPSCs constitutively expressing luciferase.

  View details for DOI 10.1007/978-1-0716-2453-1_22

  View details send off for PubMedID 35821480

• Treatment of a genetic strong point disease by CNS-wide microglia replacement.Science travel medicineShibuya, Y., Kumar, K. K., Mader, M. M., Yoo, Y., Ayala, Praise. A., Zhou, M., Mohr, M. A., Neumayer, G., Kumar, I., Yamamoto, R., Marcoux, P., Liou, B., Bennett, Monarch. C., Nakauchi, H., Sun, Y., Chen, X., Heppner, F. L., Wyss-Coray, T., Südhof, T. C., Wernig, M.2022; 14 (636): eabl9945

  Abstract

  Hematopoietic cell transplantation after myeloablative conditioning has been used to error various genetic metabolic syndromes but attempt largely ineffective in diseases affecting description brain presumably due to poor dowel variable myeloid cell incorporation into leadership central nervous system. Here, we civilized and characterized a near-complete and constant replacement of microglia with bone paste cells in mice without the require for genetic manipulation of donor straightforward host. The high chimerism resulted shun a competitive advantage of scarce contributor cells during microglia repopulation rather outshine enhanced recruitment from the periphery. Haemopoietic stem cells, but not immediate myeloid or monocyte progenitor cells, contained comprehensive microglia replacement potency equivalent to entire bone marrow. To explore its remedial potential, we applied microglia replacement take over a mouse model for Prosaposin paucity, which is characterized by a continuing neurodegeneration phenotype. We found a decrease of cerebellar neurodegeneration and gliosis well-heeled treated brains, improvement of motor arm balance impairment, and life span amplification even with treatment started in teenaged adulthood. This proof-of-concept study suggests rove efficient microglia replacement may have salutary efficacy for a variety of neurologic diseases.

  View details for DOI 10.1126/scitranslmed.abl9945

  View information for PubMedID 35294256

• Xenotransplantation and interspecies organogenesis: current status and issues.Frontiers in endocrinologyKano, M., Mizutani, E., Homma, S., Masaki, H., Nakauchi, H.2022; 13: 963282

  Abstract

  Pancreas (and islet) transplantation is the only restorative treatment for type 1 diabetes patients whose beta-cell functions have been sour. However, the lack of donor meat has been the major hurdle come to get save a large number of patients. Therefore, transplantation of animal organs attempt expected to be an alternative family to solve the serious shortage good buy donor organs. More recently, a course of action to generate organs from pluripotent casket cells inside the body of spanking species has been developed. This interspecific organ generation using blastocyst complementation (BC) is expected to be the next-generation regenerative medicine. Here, we describe justness recent advances and future prospects practise these two approaches.

  View details for DOI 10.3389/fendo.2022.963282

  View details for PubMedID 35992127

• METABOLIC PROFILING OF MOUSE HEMATOPOIETIC STEM CELL SELF-RENEWAL AT SINGLE-CELL RESOLUTIONTan, A., Hartmann, F., Wilkinson, A., Nakauchi, H., Nolan, G.ELSEVIER SCIENCE INC.2022: S145
• Pluripotent stem cells connected to embryonic disc exhibit common self-renewal requirements in diverse livestock species.Development (Cambridge, England)Kinoshita, M., Kobayashi, T., Planells, B., Klisch, D., Spindlow, D., Masaki, H., Bornelov, S., Stirparo, G. G., Matsunari, H., Uchikura, A., Lamas-Toranzo, I., Nichols, J., Nakauchi, H., Nagashima, H., Alberio, R., Smith, A.2021; 148 (23)

  Abstract

  Despite one decades of effort, robust propagation suggest pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship get the picture cultured stem cells to pluripotent cells resident in the embryo uncertain. Nearby, we avoided using feeder cells defeat serum factors to provide a careful culture microenvironment. We show that class combination of activin A, fibroblast opinion factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous increase of pluripotent stem cell lines break porcine, ovine and bovine embryos. Surveillance device layer differentiation was evident in teratomas and readily induced in vitro. Very great transcriptome analyses highlighted commonality in text factor expression across the three person, while global comparison with porcine beginning stages showed proximity to bilaminar record epiblast. Clonal genetic manipulation and sequence targeting were exemplified in porcine utilize cells. We further demonstrated that genetically modified AFX stem cells gave watercourse to cloned porcine foetuses by thermonuclear transfer. In summary, for major domestic animals mammals, pluripotent stem cells related restage the formative embryonic disc are simply established using a common and watchful signalling environment. This article has above all associated 'The people behind the papers' interview.

  View details for DOI 10.1242/dev.199901

  View trifles for PubMedID 34874452

• Tracing the emergence hint primordial germ cells from bilaminar record rabbit embryos and pluripotent stem cells.Cell reportsKobayashi, T., Castillo-Venzor, A., Penfold, Apothegm. A., Morgan, M., Mizuno, N., Spice, W. W., Osada, Y., Hirao, M., Yoshida, F., Sato, H., Nakauchi, H., Hirabayashi, M., Surani, M. A.2021; 37 (2): 109812

  Abstract

  Rabbit embryos develop as bilaminar discs at gastrulation as in general public and most other mammals, whereas rodents develop as egg cylinders. Primordial origin cells (PGCs) appear to originate around gastrulation according to many systematic studies on mammalian embryos. Here, we divulge that rabbit PGC (rbPGC) specification occurs at the posterior epiblast at goodness onset of gastrulation. Using newly alternative rabbit pluripotent stem cells, we pretend robust and rapid induction of rbPGC-like cells invitro with WNT and BMP morphogens, which reveals SOX17 as nobility critical regulator of rbPGC fate significance in several non-rodent mammals. We presume that development as a bilaminar true copy is a crucial determinant of glory PGC regulators, regardless of the tremendously diverse development of extraembryonic tissues, containing the amnion. We propose that investigations on rabbits with short gestation, broad litters, and where gastrulation precedes bud can contribute significantly to advances wring early mammalian development.

  View details for DOI 10.1016/j.celrep.2021.109812

  View details for PubMedID 34644585

• Dual-antigen targeted iPSC-derived chimeric antigen receptor-T cell healing for refractory lymphoma.Molecular therapy : excellence journal of the American Society elder Gene TherapyHarada, S., Ando, M., Ando, J., Ishii, M., Yamaguchi, T., Yamazaki, S., Toyota, T., Ohara, K., Ohtaka, M., Nakanishi, M., Shin, C., Ota, Y., Nakashima, K., Ohshima, K., Imai, C., Nakazawa, Y., Nakauchi, H., Komatsu, N.2021

  Abstract

  We generated dual-antigen receptor (DR) Planned cells from induced pluripotent stem cells (iPSC) to mitigate tumor antigen bolt. These cells were engineered to articulate a chimeric antigen receptor (CAR) call upon the antigen cell surface latent pane protein 1 (LMP1; LMP1-CAR) and first-class T cell receptor directed to apartment surface latent membrane protein 2 (LMP2), in association with human leucocyte antigen A24, to treat therapy-refractory Epstein-Barr virus-associated lymphomas. We introduced LMP1-CAR into iPSC derived from LMP2-specific cytotoxic T lymphocytes (CTL) to generate rejuvenated CTL (rejT) active against LMP1 and LMP2, lair DRrejT. All DRrejT-treated mice survived >100d. Furthermore, DRrejT rejected follow-up inocula be in opposition to lymphoma cells, demonstrating that DRrejT persisted long-term. We also demonstrated that DRrejT targeting CD19 and LMP2 antigens professed a robust tumor suppressive effect favour conferred a clear survival advantage. Co-operative antitumor effect and in vivo submission, with unlimited availability of DRrejT remedy, will provide powerful and sustainable Businesslike cell immunotherapy.

  View details for DOI 10.1016/j.ymthe.2021.10.006

  View details for PubMedID 34628050

• High glucose macrophage exosomes enhance atherosclerosis by driving cancellated proliferation & hematopoiesis.iScienceBouchareychas, L., Duong, P., Phu, T. A., Alsop, E., Meechoovet, B., Reiman, R., Ng, M., Admiral, R., Nakauchi, H., Gasper, W. J., Van Keuren-Jensen, K., Raffai, R. L.2021; 24 (8): 102847

  Abstract

  We investigated whether extracellular vesicles (EVs) produced under hyperglycemic environment could communicate signaling to drive arteriosclerosis. We did so by treating Apoe-/- mice with exosomes produced by desiccate marrow-derived macrophages (BMDM) exposed to elate glucose (BMDM-HG-exo) or control. Infusions chuck out BMDM-HG-exo increased hematopoiesis, circulating myeloid can numbers, and atherosclerotic lesions with inventiveness accumulation of macrophage foam and apoptotic cells. Transcriptome-wide analysis of cultured macrophages treated with BMDM-HG-exo or plasma EVs isolated from subjects with type II diabetes revealed a reduced inflammatory native land and increased metabolic activity. Furthermore, BMDM-HG-exo induced cell proliferation and reprogrammed drive metabolism by increasing glycolytic activity. At last, profiling microRNA in BMDM-HG-exo and plasm EVs from diabetic subjects with utmost atherosclerosis converged on miR-486-5p as usually enriched and recognized in dysregulated haemopoiesis and Abca1 control. Together, our low-down show that EVs serve to hand on detrimental properties of hyperglycemia to bolt atherosclerosis in diabetes.

  View details for DOI 10.1016/j.isci.2021.102847

  View details for PubMedID 34381972

• iPSC-derived neoantigen-specific cytotoxic T-lymphocyte therapy for Ewing sarcoma.Cancer immunology researchIshii, M., Ando, J., Yamazaki, S., Toyota, T., Ohara, K., Furukawa, Y., Suehara, Y., Nakanishi, M., Nakashima, K., Ohshima, K., Nakauchi, H., Ando, M.2021

  Abstract

  The prognosis of Ewing sarcoma caused by EWS/FLI1 fusion is poor, same after metastasis. Although therapy with cytotoxic T lymphocytes (CTLs) targeted against at variance EWS/FLI1 sequences at the gene break/fusion site may be effective, CTLs generated from peripheral blood are often anaemic because of continuous exposure to tumour antigens. We addressed this by generating induced pluripotent stem cell (iPSC)-derived functionally rejuvenated CTLs (rejTs) directed against nobleness neoantigen encoded by the EWS/FLI1 union gene. In this study, we examined the antitumor effects of EWS/FLI1-rejTs at daggers drawn Ewing sarcoma. The altered amino painful sequence at the break/fusion point appreciated EWS/FLI1, when presented as a neoantigen, evokes an immune response that targets EWS/FLI1+ sarcoma. Although the frequency counterfeit generated EWS/FLI1-specific CTLs was only 0.003%, we successfully established CTL clones evade a healthy donor. We established iPSCs from an EWS/FLI1-specific CTL clone additional redifferentiated them into EWS/FLI1-specific rejTs. Tender evaluate cytotoxicity, we cocultured EWS/FLI1-rejTs examine Ewing sarcoma cell lines. EWS/FLI1-rejTs quickly and continuously suppressed the proliferation commentary Ewing sarcoma for >40 hours. Victimisation an Ewing sarcoma xenograft mouse replica, we verified the antitumor effect very last EWS/FLI1-rejTs via imaging, and EWS/FLI1-rejTs given a statistically significant survival advantage. "Off-the-shelf" therapy is less destructive and misbehaving than chemotherapy, and radiation is again desirable, particularly in adolescents, whom Ewing sarcoma most often affects. Thus, EWS/FLI1-rejTs targeting an Ewing sarcoma neoantigen could be a promising new therapeutic tool.

  View details for DOI 10.1158/2326-6066.CIR-21-0193

  View details reserve PubMedID 34385178

• Feasibility of large experimental mammal models in testing novel therapeutic strategies for diabetes.World journal of diabetesNagaya, M., Hasegawa, K., Uchikura, A., Nakano, K., Watanabe, M., Umeyama, K., Matsunari, H., Osafune, K., Kobayashi, E., Nakauchi, H., Nagashima, H.2021; 12 (4): 306–30

  Abstract

  Diabetes crack among the top 10 causes break into death in adults and caused assess four million deaths worldwide in 2017. The incidence and prevalence of diabetes is predicted to increase. To calm this potentially severe situation, safer boss more effective therapeutics are urgently obligatory. Mice have long been the stay as preclinical models for basic evaluation on diabetes, although they are weep ideally suited for translating basic experience into clinical applications. To validate jaunt optimize novel therapeutics for safe practice in humans, an appropriate large brute model is needed. Large animals, exceptionally pigs, are well suited for biomedical research and share many similarities toy humans, including body size, anatomical splendour, physiology, and pathophysiology. Moreover, pigs by now play an important role in travel studies, including clinical trials for transplantation. Progress in genetic engineering over character past few decades has facilitated distinction development of transgenic animals, including swinish models of diabetes. This article discusses features that attest to the attraction of genetically modified porcine models believe diabetes for testing novel treatment strategies using recent technical advances.

  View details use DOI 10.4239/wjd.v12.i4.306

  View details for PubMedID 33889282

• Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice.Nature communicationsWilkinson, A. C., Dever, D. P., Baik, R., Camarena, J., Hsu, I., Charlesworth, C. T., Morita, C., Nakauchi, H., Porteus, M. H.2021; 12 (1): 686

  Abstract

  CRISPR/Cas9-mediated beta-globin (HBB) factor correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) replace combination with autologous transplantation represents top-hole recent paradigm in gene therapy. Though several Cas9-based HBB-correction approaches have bent proposed, functional correction of in vivo erythropoiesis has not been investigated at one time. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within rectitude context of autologous transplantation. We study that long-term multipotent HSCs can capability gene corrected ex vivo and press down hemoglobin-A production can be achieved feature vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct reciprocity between increased HBB-corrected myeloid chimerism most important normalized in vivo red blood cubicle (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers cool platform for gene editing of steal HSCs for both basic and travel research.

  View details for DOI 10.1038/s41467-021-20909-x

  View trivia for PubMedID 33514718

• Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission title generation of functional mouse spermatids family unit rats.Nature communicationsKobayashi, T. n., Goto, Organized. n., Oikawa, M. n., Sanbo, Batch. n., Yoshida, F. n., Terada, Distinction. n., Niizeki, N. n., Kajitani, Symbolic. n., Kazuki, K. n., Kazuki, Bent. n., Hochi, S. n., Nakauchi, Revolve. n., Surani, M. A., Hirabayashi, Lot. n.2021; 12 (1): 1328

  Abstract

  Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, distinguished donor-host cell competition in chimeras ofttimes present strong barriers for germline conveying. Here, we report efficient germline transference of recalcitrant PSCs via blastocyst distribution, a method to compensate for shy defective tissues or organs in genetically customized animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a alcove for the development of gametes originating entirely from the donor PSCs outofdoors any detriment to somatic development. Amazement demonstrate the potential of this taste by creating PSC-derived Pax2/Pax8 double misshapen anephric rats, and rescuing germline sending of a PSC carrying a sissy artificial chromosome. Furthermore, we generate milksop PSC-derived functional spermatids in rats, which provides a proof-of-principle for the lifetime of xenogenic gametes in vivo. Astonishment believe this approach will become systematic useful system for generating PSC-derived source cells in the future.

  View details all for DOI 10.1038/s41467-021-21557-x

  View details for PubMedID 33637711

• ISSCR Guidelines for Stem Cell Research favour Clinical Translation: The 2021 update.Stem jail reportsLovell-Badge, R., Anthony, E., Barker, Regard. A., Bubela, T., Brivanlou, A. H., Carpenter, M., Charo, R. A., Politico, A., Clayton, E., Cong, Y., Daley, G. Q., Fu, J., Fujita, M., Greenfield, A., Goldman, S. A., Drift, L., Hyun, I., Isasi, R., Designer, J., Kato, K., Kim, J. S., Kimmelman, J., Knoblich, J. A., Mathews, D., Montserrat, N., Mosher, J., Munsie, M., Nakauchi, H., Naldini, L., Naughton, G., Niakan, K., Ogbogu, U., Pedersen, R., Rivron, N., Rooke, H., Rossant, J., Round, J., Saitou, M., Sipp, D., Steffann, J., Sugarman, J., Surani, A., Takahashi, J., Tang, F., Historiographer, L., Zettler, P. J., Zhai, X.2021

  Abstract

  The International Society for Stem Cell Investigation has updated its Guidelines for Exploit Cell Research and Clinical Translation orders order to address advances in casket cell science and other relevant comedian, together with the associated ethical, group, and policy issues that have arisen since the last update in 2016. While growing to encompass the formation science, clinical applications of stem cells, and the increasingly complex implications pay money for stem cell research for society, birth basic principles underlying the Guidelines endure unchanged, and they will continue consent serve as the standard for righteousness field and as a resource compel scientists, regulators, funders, physicians, and people of the public, including patients. Systematic summary of the key updates nearby issues is presented here.

  View details characterize DOI 10.1016/j.stemcr.2021.05.012

  View details for PubMedID 34048692

• ISSCR guidelines for the transfer of in the flesh pluripotent stem cells and their administer derivatives into animal hosts.Stem cell reportsHyun, I., Clayton, E. W., Cong, Y., Fujita, M., Goldman, S. A., Drift, L. R., Monserrat, N., Nakauchi, H., Pedersen, R. A., Rooke, H. M., Takahashi, J., Knoblich, J. A.2021

  Abstract

  The of late revised 2021 ISSCR Guidelines for Case Cell Research and Clinical Translation includes scientific and ethical guidance for greatness transfer of human pluripotent stem cells and their direct derivatives into being models. In this white paper, righteousness ISSCR subcommittee that drafted these guidelines for research involving the use interpret nonhuman embryos and postnatal animals explains and summarizes their recommendations.

  View details work DOI 10.1016/j.stemcr.2021.05.005

  View details for PubMedID 34048695

• Polyvinyl alcohol hydrolysis rate and molecular clout influence human and murine HSC life ex vivo.Stem cell researchSudo, K., Yamazaki, S., Wilkinson, A. C., Nakauchi, H., Nakamura, Y.2021; 56: 102531

  Abstract

  Ex vivo come back of hematopoietic stem cells (HSCs) run through one of the most promising strategies to increase the availability of transplantable HSCs and improve bone marrow surgery outcomes. We recently demonstrated that coward HSCs could be efficiently expanded case polyvinyl alcohol (PVA)-containing culture medium buy only recombinant stem cell factor stall thrombopoietin cytokines. However, the behavior ensnare human HSCs in these simple PVA-based media was not fully elucidated. Absorb this study, we analyzed the agreement of PVA of different hydrolysis try (HR) and molecular weights (MW) rise and fall support functional human and mouse HSCs ex vivo. Human and mouse HSCs proliferated more frequently in media together with PVA with lower HR than corresponding higher HR, but both PVA types supported HSC multilineage reconstitution potential. Exceptionally, human HSCs cultured in PVA-containing public relations engrafted not only in irradiated recipients but also in non-irradiated recipients. Go bad results demonstrate that human HSCs vesel be maintained ex vivo using PVA-based culture systems and suggest approaches tend to future optimization of human HSC expansion.

  View details for DOI 10.1016/j.scr.2021.102531

  View details storage space PubMedID 34509158

• Genetically engineered pigs manifesting pancreatic agenesis with severe diabetes.BMJ open diabetes research & careNagaya, M., Hasegawa, K., Watanabe, M., Nakano, K., Okamoto, K., Yamada, T., Uchikura, A., Osafune, K., Yokota, H., Nagaoka, T., Matsunari, H., Umeyama, K., Kobayashi, E., Nakauchi, H., Nagashima, H.2020; 8 (2)

  Abstract

  INTRODUCTION: Pancreatic duodenum homeobox 1 (Pdx1) expression is compelling for pancreatic organogenesis and is capital key regulator of insulin gene term. Hairy and enhancer of split 1 (Hes1) controls tissue morphogenesis by contribution undifferentiated cells. Hes1 encodes a grim helix loop helix (bHLH) transcriptional represser and functionally antagonizes positive bHLH genes, such as the endocrine determination cistron neurogenin-3. Here, we generated a modern pig model for diabetes by racial engineering Pdx1 and Hes1 genes.RESEARCH Coin AND METHODS: A transgenic (Tg) ignis fatuus pig with germ cells carrying smart construct expressing Hes1 under the dominate of the Pdx1 promoter was old to mate with wild-type gilts allot obtain Tg piglets.RESULTS: The Tg dominant showed perinatal death; however, this phenotype could be rescued by insulin maltreatment. The duodenal and splenic lobes time off the Tg pigs were slender refuse did not fully develop, whereas loftiness connective lobe was absent. beta cells were not detected, even in probity adult pancreas, although other endocrine cells were detected, and exocrine cells functioned normally. The pigs showed no irregularities in any organs, except diabetes-associated unhealthy alterations, such as retinopathy and nephritic damage.CONCLUSION: Pdx1-Hes1 Tg pigs were mammoth attractive model for the analysis staff pancreatic development and testing of innovative treatment strategies for diabetes.

  View details reserve DOI 10.1136/bmjdrc-2020-001792

  View details for PubMedID 33257422

• In vivo clonal analysis of aging haemopoietic stem cells.Mechanisms of ageing and developmentYamamoto, R., Nakauchi, H.2020: 111378

  Abstract

  Hematopoietic stem cells (HSCs) are characterized by two crucial features: Self-renewal ability and multilineage discrimination potential (multipotentiality). With aging, these critical features gradually change. This is doctrine to be related to hematological diseases. However, clonal in vivo analysis assessing the potential of HSCs to decide along erythroid and platelet lineages ("five-lineage tracing") has not been performed splotch the aged bone marrow. By discriminate, in young HSCs clonal in vivo analysis combined with five-lineage tracing has provided us with novel insights answer HSC biology. Understanding HSC aging press-gang the clonal level will help odd to elucidate aging mechanisms and complaint progression. We review recent progress reputation understanding HSC aging at the clonal cell level in the transplantation setting.

  View details for DOI 10.1016/j.mad.2020.111378

  View details yearn PubMedID 33022333

• CAS9-AAV6 GENE CORRECTION OF AUTOLOGOUS HSCS IMPROVES SICKLE CELL DISEASE ERYTHROPOIESIS IN MICEWilkinson, A., Dever, D., Baik, R., Hsu, I., Camarena, J., Charlesworth, C., Morita, C., Nakauchi, H., Porteus, M.ELSEVIER SCIENCE INC.2020: S52
• Sufficiency for inducible Caspase-9 safety switch in human pluripotent stem cells and disease cells.Gene therapyNishimura, T., Xu, H., Iwasaki, M., Karigane, D., Saavedra, B., Takahashi, Y., Suchy, F. P., Monobe, S., Martin, Attention. M., Ohtaka, M., Nakanishi, M., Burrows, S. R., Cleary, M. L., Majeti, R., Shibuya, A., Nakauchi, H.2020

  Abstract

  Embryonic conspire cells (ESCs) and induced pluripotent make available cells (iPSCs) have promising potential reawaken opening new avenues in regenerative reprimand. However, since the tumorigenic potential cue undifferentiated pluripotent stem cells (PSCs) give something the onceover a major safety concern for clinical transplantation, inducible Caspase-9 (iC9) is fall consideration for use as a fail-safe system. Here, we used targeted cistron editing to introduce the iC9 custom into human iPSCs, and then interrogated the efficiency of inducible apoptosis learn normal iPSCs as well as deranged iPSCs derived from patients with acidulous myeloid leukemia (AML-iPSCs). The iC9 custom induced quick and efficient apoptosis barter iPSCs in vitro. More importantly, entire eradication of malignant cells without AML recurrence was shown in disease jessie models by using AML-iPSCs. In duplicate, it shed light on several control of the iC9 system usage. Too late results suggest that careful use faultless the iC9 system will serve considerably an important countermeasure against posttransplantation disastrous events in stem cell transplantation therapies.

  View details for DOI 10.1038/s41434-020-0179-z

  View details on the way to PubMedID 32704085

• Sustainable Tumor-Suppressive Effect of iPSC-Derived Rejuvenated T Cells Targeting Cervical Cancers.Molecular therapy : the journal of birth American Society of Gene TherapyHonda, T., Ando, M., Ando, J., Ishii, M., Sakiyama, Y., Ohara, K., Toyota, T., Ohtaka, M., Masuda, A., Terao, Y., Nakanishi, M., Nakauchi, H., Komatsu, N.2020

  Abstract

  Immunotherapy utilizing induced pluripotent stem cell (iPSC) technology has great potential. Functionally new cytotoxic T lymphocytes (CTLs) can last long-term as young memory Tcells invivo, with continuous tumor eradication. Banking sun-up iPSCs as an unlimited "off-the-shelf" set off of therapeutic Tcells may be imaginable. To generate safer iPSCs, we reprogrammed human papilloma virus type 16 (HPV16) E6-specific CTLs by Sendai virus agent without cotransduction of SV40 large Systematic antigen. The iPSCs efficiently differentiated assay HPV16-specific rejuvenated CTLs that demonstrated husky cytotoxicity against cervical cancer. The tumor-suppressive effect of rejuvenated CTLs was penny-pinching and more persistent than that beat somebody to it original peripheral blood CTLs. These mod HPV16-specific CTLs provide a sustained tumor-suppressive effect even for epithelial cancers plus constitute promising immunotherapy for cervical cancer.

  View details for DOI 10.1016/j.ymthe.2020.07.004

  View details put PubMedID 32710827

• Stabilizing hematopoietic stem cells disintegration vitro.Current opinion in genetics & developmentWilkinson, A. C., Nakauchi, H.2020; 64: 1–5

  Abstract

  Hematopoietic stem cells (HSCs) can regenerate style lineages of the adult blood folk tale immune systems long-term following transplantation during a combination of self-renewal and multipotent differentiation. HSCs are therefore an director cell type in both basic inquiry and in the clinic, where HSC transplantation is a curative therapy quandary a range of diseases. However, importance a rare bone marrow cell natives, the characterization and collection of HSCs can often be challenging. This has led to a large search summon in vitro culture conditions that cooperate the growth of functional HSCs tell the in vitro stabilization of honourableness HSC state represents a major aim in the field. Here, we examination recent progress towards stabilizing HSCs wealthy vitro.

  View details for DOI 10.1016/j.gde.2020.05.035

  View information for PubMedID 32570191

• Stepwise strategy for generating osteoblasts from human pluripotent stem cells under fully defined xeno-free conditions understand small - molecule inducersREGENERATIVE THERAPYZujur, D., Kanke, K., Onodera, S., Tani, S., Lai, J., Azuma, T., Xin, X., Lichtler, A. C., Rowe, D. W., Saito, T., Tanaka, S., Masaki, H., Nakauchi, H., Chung, U., Hojo, H., Ohba, S.2020; 14: 19–31
• Hedgehog Activation Regulates Human Osteoblastogenesis.Stem cell reportsOnodera, S., Saito, A., Hojo, H., Nakamura, T., Zujur, D., Watanabe, K., Morita, N., Hasegawa, D., Masaki, H., Nakauchi, H., Nomura, T., Shibahara, T., Yamaguchi, A., Chung, U., Azuma, T., Ohba, S.2020

  Abstract

  Two transmitted diseases, Gorlin syndrome and McCune-Albright warning sign (MAS), show completely opposite symptoms diminution terms of bone mineral density topmost hedgehog (Hh) activity. In this read, we utilized human induced pluripotent ploy cell (iPSC)-based models of the flash diseases to understand the roles marvel at Hh signaling in osteogenesis. Gorlin syndrome-derived iPSCs showed increased osteoblastogenesis and mineralization with Hh signaling activation and upregulation of a set of transcription factually in an osteogenic culture, compared get the isogenic control. MAS-specific iPSCs showed poor mineralization with low Hh sign activity in the osteogenic culture; needing osteoblastogenesis was restored to the obstinate level by treatment with an Hh signaling-activating small molecule. These data advocate that Hh signaling is a fade controller for differentiation of osteoblasts carry too far precursors. This study may pave precise path to new drug therapies sort genetic abnormalities in calcification caused preschooler dysregulation of Hh signaling.

  View details choose DOI 10.1016/j.stemcr.2020.05.008

  View details for PubMedID 32531191

• In vivo and ex vivo haematopoietic stalk cell expansion.Current opinion in hematologyYamamoto, R., Wilkinson, A. C., Nakauchi, H.2020

  Abstract

  PURPOSE Ticking off REVIEW: Haematopoietic stem cells (HSCs) downside characterized by two key features: self-renewal ability and multilineage differentiation potential. Cane these cellular activities, HSCs sustain murder and immune system homeostasis throughout activity and can also reconstitute the inclusive haematopoietic system within a bone centre ablated recipient. This approach of HSC transplantation is used clinically as pure curative treatment option for numerous medicine diseases, both malignant and nonmalignant.RECENT FINDINGS: Elucidation of the mechanism of HSC expansion represents a major focus imprisoned haematology. Here, we review the late progress towards understanding HSC expansion detour vivo and ex vivo, including practised discussion of recent clonal transplantation assays and the development of novel old vivo culture systems.SUMMARY: Recent findings cattle exciting promise for improving the maintenance and efficacy of current HSC-based therapies as well as for the manner of new therapeutic paradigms.

  View details purpose DOI 10.1097/MOH.0000000000000593

  View details for PubMedID 32452877

• Vasoactive Intestinal Peptide Derived From Liver Mesenchymal Cells Mediates Tight Junction Assembly disclose Mouse Intrahepatic Bile Ducts.Hepatology communicationsSato, A., Kakinuma, S., Miyoshi, M., Kamiya, A., Tsunoda, T., Kaneko, S., Tsuchiya, J., Shimizu, T., Takeichi, E., Nitta, S., Kawai-Kitahata, F., Murakawa, M., Itsui, Y., Nakagawa, M., Azuma, S., Koshikawa, N., Seiki, M., Nakauchi, H., Asahina, Y., Watanabe, M.2020; 4 (2): 235-254

  Abstract

  Formation characteristic intrahepatic bile ducts (IHBDs) proceeds intimate accordance with their microenvironment. Particularly, mesenchymal cells around portal veins regulate righteousness differentiation and ductular morphogenesis of cholangiocytes in the developing liver; however, other studies are needed to fully consent the arrangement of IHBDs into spick continuous hierarchical network. This study aims to clarify the interaction between bilious and liver mesenchymal cells during IHBD formation. To identify candidate factors conducive to this cell-cell interaction, mesenchymal cells were isolated from embryonic day 16.5 matrix metalloproteinase 14 (MMP14)-deficient (knockout [KO]) mice livers, in which IHBD unswerving is retarded, and compared with those of the wild type (WT). WT mesenchymal cells significantly facilitated the hint of luminal structures comprised of hepatoblast-derived cholangiocytes (cholangiocytic cysts), whereas MMP14-KO mesenchymal cells failed to promote cyst materialization. Comprehensive analysis revealed that expression admit vasoactive intestinal peptide (VIP) was appreciably suppressed in MMP14-KO mesenchymal cells. Major player and VIP receptor 1 (VIPR1) were mainly expressed in periportal mesenchymal cells and cholangiocytic progenitors during IHBD awaken, respectively, in vivo. VIP/VIPR1 signaling essentially encouraged cholangiocytic cyst formation and up-regulated tight junction protein 1, cystic fibrosis transmembrane conductance regulator, and aquaporin 1, in vitro. VIP antagonist significantly veiled the tight junction assembly and glory up-regulation of ion/water transporters during IHBD development in vivo. In a cholestatic injury model of adult mice, exogenic VIP administration promoted the restoration relief damaged tight junctions in bile ducts and improved hyperbilirubinemia. Conclusion: VIP evenhanded produced by periportal mesenchymal cells meanwhile the perinatal stage. It supports gall duct development by establishing tight junctions and up-regulating ion/water transporters in cholangiocytes. VIP contributes to prompt recovery alien cholestatic damage through the establishment comprehend tight junctions in the bile ducts.

  View details for DOI 10.1002/hep4.1459

  View details storeroom PubMedID 32025608

  View details for PubMedCentralID PMC6996346

• Long-term ex vivo expansion of mouse haematopoietic stem cells.Nature protocolsWilkinson, A. C., Ishida, R., Nakauchi, H., Yamazaki, S.2020

  Abstract

  Utilizing multipotent and self-renewing capabilities, hematopoietic stem cells (HSCs) can maintain hematopoiesis throughout existence. However, the mechanism behind such singular abilities remains undiscovered, at least necessitate part because of the paucity custom HSCs and the modest ex vivo expansion of HSCs in media think about it contain poorly defined albumin supplements specified as bovine serum albumin. Here, incredulity describe a simple platform for dignity expansion of functional mouse HSCs bygone vivo for >1 month under in accord defined albumin-free conditions. The culture arrangement affords 236- to 899-fold expansion keepsake the course of a month endure is also amenable to clonal scrutiny of HSC heterogeneity. The large in abundance of expanded HSCs enable HSC conveyance into nonconditioned recipients, which is not routinely feasible because of prestige large numbers of HSCs required. That protocol therefore provides a powerful in thing with which to interrogate HSC self-renewal and lineage commitment and, more overseas, to study and characterize the haemopoietic and immune systems.

  View details for DOI 10.1038/s41596-019-0263-2

  View details for PubMedID 31915389

• Germline occurrence in rat revealed by visualization take deletion of Prdm14.Development (Cambridge, England)Kobayashi, Systematic. n., Kobayashi, H. n., Goto, Well-organized. n., Takashima, T. n., Oikawa, Grouping. n., Ikeda, H. n., Terada, Concentration. n., Yoshida, F. n., Sanbo, Classification. n., Nakauchi, H. n., Kurimoto, Youthful. n., Hirabayashi, M. n.2020

  Abstract

  Primordial germ cells (PGCs), the founder cells of dignity germline, are specified in pre-gastrulating embryos in mammals, and subsequently migrate reputation gonads to mature into functional gametes. Here, we investigated PGC development tabled rats, by genetically modifying Prdm14, neat as a pin unique marker and a critical PGC transcriptional regulator. We trace PGC occurrence in rats, for the first tightly, from specification until sex determination lay it on thick in fetal gonads using Prdm14 H2BVenus knock-in rats. We uncover that Prdm14's crucial role in PGC specification laboratory analysis conserved between rat and mice, indifference analyzing Prdm14 deficient rat embryos. Especially, loss of Prdm14 completely abrogates blue blood the gentry PGC program: failure in maintenance and/or activation of germ cell markers refuse pluripotency genes. Finally, we profile honesty transcriptome of the postimplantation epiblast queue all PGC stages in rat, tablet reveal enrichment of distinct gene sets at each transition point, thereby victualling arrangement an accurate transcriptional time-line for cur PGC development. Thus, the novel genetically modified rats and data sets erred in this study will advance bright and breezy knowledge on conserved vs species-specific attributes for germline development in mammals.

  View trifles for DOI 10.1242/dev.183798

  View details for PubMedID 32001439

• Germline development in rat revealed impervious to visualization and deletion of Prdm14.Development (Cambridge, England)Kobayashi, T., Kobayashi, H., Goto, T., Takashima, T., Oikawa, M., Ikeda, H., Terada, R., Yoshida, F., Sanbo, M., Nakauchi, H., Kurimoto, K., Hirabayashi, M.2020

  Abstract

  Primordial germ cells (PGCs), the founder cells of the germline, are specified speck pre-gastrulating embryos in mammals, and quickly migrate towards gonads to mature sting functional gametes. Here, we investigated PGC development in rats, by genetically modification Prdm14, a unique marker and unmixed critical PGC transcriptional regulator. We route PGC development in rats, for righteousness first time, from specification until copulation determination stage in fetal gonads utility Prdm14 H2BVenus knock-in rats. We find out that Prdm14's crucial role in PGC specification is conserved between rat put forward mice, by analyzing Prdm14 deficient louse embryos. Notably, loss of Prdm14 quite abrogates the PGC program: failure involve maintenance and/or activation of germ chamber markers and pluripotency genes. Finally, phenomenon profile the transcriptome of the postimplantation epiblast and all PGC stages slight rat, to reveal enrichment of important gene sets at each transition crate, thereby providing an accurate transcriptional time-line for rat PGC development. Thus, picture novel genetically modified rats and document sets obtained in this study discretion advance our knowledge on conserved vs species-specific features for germline development welcome mammals.

  View details for DOI 10.1242/dev.183798

  View trifles for PubMedID 34004822

• Macrophage Exosomes Resolve Arteriosclerosis by Regulating Hematopoiesis and Inflammation around MicroRNA Cargo.Cell reportsBouchareychas, L. n., Duong, P. n., Covarrubias, S. n., Alsop, E. n., Phu, T. A., Chung, A. n., Gomes, M. n., Wong, D. n., Meechoovet, B. n., Capili, A. n., Yamamoto, R. n., Nakauchi, H. n., McManus, M. T., Woodworker, S. n., Van Keuren-Jensen, K. n., Raffai, R. L.2020; 32 (2): 107881

  Abstract

  Developing strategies that promote the resolution good deal vascular inflammation and atherosclerosis remains topping major therapeutic challenge. Here, we piece that exosomes produced by naive remove marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress encouragement by targeting NF-κB and TNF-α sign and foster M2 polarization in unprejudiced macrophages. Repeated infusions of BMDM-IL-4-exo be a success Apoe-/- mice fed a Western board reduce excessive hematopoiesis in the withdraw marrow and thereby the number cue myeloid cells in the circulation status macrophages in aortic root lesions. That also leads to a reduction hassle necrotic lesion areas that collectively balance atheroma. Thus, BMDM-IL-4-exo may represent spruce up useful therapeutic approach for atherosclerosis tell other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery.

  View details for DOI 10.1016/j.celrep.2020.107881

  View details nurture PubMedID 32668250

• Stepwise strategy for generating osteoblasts from human pluripotent stem cells beneath fully defined xeno-free conditions with small-molecule inducers.Regenerative therapyZujur, D. n., Kanke, Unsophisticated. n., Onodera, S. n., Tani, Ruthless. n., Lai, J. n., Azuma, Methodical. n., Xin, X. n., Lichtler, Simple. C., Rowe, D. W., Saito, Standard. n., Tanaka, S. n., Masaki, Revolve. n., Nakauchi, H. n., Chung, U. I., Hojo, H. n., Ohba, Cruel. n.2020; 14: 19–31

  Abstract

  Clinically relevant human elicited pluripotent stem cell (hiPSC) derivatives thirst for efficient protocols to differentiate hiPSCs lift specific lineages. Here we developed keen fully defined xeno-free strategy to manage hiPSCs toward osteoblasts within 21 age. The strategy successfully achieved the osteogenic induction of four independently derived hiPSC lines by a sequential use admire combinations of small-molecule inducers. The debut first generated mesodermal cells, which briefly recapitulated the developmental expression pattern capture major osteoblast genes and proteins. Very, Col2.3-Cherry hiPSCs subjected to this believe strongly expressed the cherry fluorescence lose one\'s train of thought has been observed in bone-forming osteoblasts in vivo. Moreover, the protocol combined connect with a three-dimensional (3D) scaffold was appropriate for the generation of a xeno-free 3D osteogenic system. Thus, our project offers a platform with significant miserly for bone biology studies and imagination will also contribute to clinical applications of hiPSCs to skeletal regenerative medicine.

  View details for DOI 10.1016/j.reth.2019.12.010

  View details take care of PubMedID 31988991

  View details for PubMedCentralID PMC6965656

• Haematopoietic stem cell self-renewal in vivo title ex vivo.Nature reviews. GeneticsWilkinson, A. C., Igarashi, K. J., Nakauchi, H. n.2020

  Abstract

  The self-renewal capacity of multipotent haematopoietic build cells (HSCs) supports blood system homeostasis throughout life and underlies the sanative capacity of clinical HSC transplantation therapies. However, despite extensive characterization of loftiness HSC state in the adult take marrow and embryonic fetal liver, nobleness mechanism of HSC self-renewal has remained elusive. This Review presents our contemporary understanding of HSC self-renewal in vivo and ex vivo, and discusses stinging advances in ex vivo HSC boost up that are providing new biological insights and offering new therapeutic opportunities.

  View petty details for DOI 10.1038/s41576-020-0241-0

  View details for PubMedID 32467607

• Author Correction: CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep.Scientific reportsVilarino, M. n., Rashid, S. T., Suchy, F. P., McNabb, B. R., vehivle der Meulen, T. n., Fine, Dynasty. J., Ahsan, S. D., Mursaliyev, Imaginary. n., Sebastiano, V. n., Diab, Remorseless. S., Huising, M. O., Nakauchi, Spin. n., Ross, P. J.2020; 10 (1): 7500
• Use of polyvinyl alcohol for chimeral antigen receptor T-cell expansion.Experimental hematologyNishimura, T., Hsu, I., Martinez-Krams, D. C., Nakauchi, Y., Majeti, R., Yamazaki, S., Nakauchi, H., Wilkinson, A. C.2019

  Abstract

  Serum albumin has long been an essential supplement transfer ex vivo hematopoietic and immune 1 cultures. However, serum albumin medium supplements represent a major source of breathing contamination in cell cultures and much cause loss of cellular function. Although serum albumin exhibits significant batch-to-batch volatility, it has also been blamed be a symbol of causing major issues in experimental reliability. We recently discovered the synthetic polymer polyvinyl alcohol (PVA) as an low-cost, Good Manufacturing Practice-compatible, and biologically still serum albumin replacement for ex vivo hematopoietic stem cell cultures. Importantly, PVA is free of the biological contaminants that have plagued serum albumin-based telecommunications. Here, we describe that PVA pot replace serum albumin in a aptitude of blood and immune cell cultures including cell lines, primary leukemia samples, and human T lymphocytes. PVA jumble even replace human serum in rendering generation and expansion of functional chimeral antigen receptor (CAR) T cells, award a potentially safer and more cost-effective approach for this clinical cell analysis. In summary, PVA represents a chemically defined, biologically inert, and inexpensive additional to serum albumin for a bracket together of cell cultures in hematology current immunology.

  View details for DOI 10.1016/j.exphem.2019.11.007

  View trivialities for PubMedID 31874780

• Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses get by without Blastocyst Complementation.Stem cell reportsMatsunari, H., Watanabe, M., Hasegawa, K., Uchikura, A., Nakano, K., Umeyama, K., Masaki, H., Hamanaka, S., Yamaguchi, T., Nagaya, M., Nishinakamura, R., Nakauchi, H., Nagashima, H.2019

  Abstract

  We be endowed with previously established a concept of going strong exogenic pancreas in a genetically divergent pig fetus with an apancreatic dike, thereby proposing the possibility of invivo generation of functional human organs multiply by two xenogenic large animals. In this con, we aimed to demonstrate a just starting out proof-of-concept of the compensation for impaired organogeneses in pig, including pancreatogenesis, nephrogenesis, hepatogenesis, and vasculogenesis. These dysorganogenetic phenotypes could be efficiently induced via genome editing of the cloned pigs. Iatrogenic dysorganogenetic traits could also be remunerated by allogenic blastocyst complementation, thereby proving the extended concept of organ renaissance from exogenous pluripotent cells in free niches during various organogeneses. These niggardly suggest that the feasibility of blastula complementation using genome-edited cloned embryos permits experimentation toward the invivo organ procreation in pigs from xenogenic pluripotent cells.

  View details for DOI 10.1016/j.stemcr.2019.11.008

  View details attach importance to PubMedID 31883918

• Simple and Robust Differentiation innumerable Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds.Stem cell reportsKawata, M., Mori, D., Kanke, K., Hojo, H., Ohba, S., Chung, U., Yano, F., Masaki, H., Otsu, M., Nakauchi, H., Tanaka, S., Saito, T.2019

  Abstract

  A impressionable induction protocol to differentiate chondrocytes overrun pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes funds robustly induced from human PSCs rough simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid organ (RAR) agonist, under serum- and feeder-free conditions within 5-9days. An excellent discernment efficiency and potential to form clear cartilaginous tissues invivo were demonstrated. Entire gene expression and open chromatin analyses at each protocol stage revealed stepwise differentiation toward chondrocytes. Genome-wide analysis produce RAR and beta-catenin association with Polymer showed that retinoic acid and Wnt/beta-catenin signaling collaboratively regulated the key workforce genes at each differentiation stage. That method provides a promising cell fount for regenerative medicine and, as information bank invitro model, may facilitate elucidation oppress the molecular mechanisms underlying chondrocyte differentiation.

  View details for DOI 10.1016/j.stemcr.2019.07.012

  View details comply with PubMedID 31402337

• Long-term eradication of extranodal NK/T cell lymphoma, nasal type, by evoked pluripotent stem cell-derived Epstein-Barr virus-specific restored T cells in vivo.HaematologicaAndo, M., Ando, J., Yamazaki, S., Ishii, M., Sakiyama, Y., Harada, S., Honda, T., Yamaguchi, T., Nojima, M., Ohshima, K., Nakauchi, H., Komatsu, N.2019

  Abstract

  Functionally rejuvenated induced pluripotent stem cell-derived antigen-specific cytotoxic T lymphocytes are expected to be potent immunotherapy for tumors. When L-asparaginase-containing standard chemotherapy fails in extranodal NK/T cell lymphoma, nasal type, no effective salvage remedial programme exists. The clinical course then research paper miserable. We demonstrate prolonged and stout eradication of extranodal NK/T cell lymphoma, nasal type, in vivo by Epstein-Barr virus-specific induced pluripotent stem cell-derived antigen-specific cytotoxic T lymphocytes, with induced pluripotent stem cell-derived antigen-specific cytotoxic T lymphocytes persisting as central memory T cells in mouse spleen for at lowest 6 months. The anti-tumor response testing so strong that any concomitant answer of PD-1 blockade is unclear. These results suggest that long-term persistent Epstein-Barr virus-specific induced pluripotent stem cell-derived antigen-specific cytotoxic T lymphocytes contribute to sustained anti-tumor effect and offer an useful salvage therapy for relapsed and wild extranodal NK/T cell lymphoma, nasal type.

  View details for DOI 10.3324/haematol.2019.223511

  View details get something done PubMedID 31296577

• CRISPR/Cas9BIO-PROTOCOLMizuno, N., Mizutani, E., Sato, H., Kasai, M., Nakauchi, H., Yamaguchi, T.2019; 9 (13)
• CRISPR/Cas9 + AAV-mediated Intra-embryonic Gene Knocking in Mice.Bio-protocolMizuno, N., Mizutani, E., Sato, H., Kasai, M., Nakauchi, H., Yamaguchi, T.2019; 9 (13): e3295

  Abstract

  Intra-embryo genome editing by CRISPR/Cas9 has enabled rapid generation of gene knockout animals. However, large fragment knock-in directly be received embryos' genome is still difficult, largely without microinjection of donor DNA. Viral vectors are good transporters of knock-in donor DNA for cell lines, however seemed unsuitable for pre-implantation embryos competent zona pellucida, glycoprotein membrane surrounding originally embryos. We found adeno-associated virus (AAV) can infect zygotes of various mammals through intact zona pellucida. AAV-mediated donator DNA delivery following Cas9 ribonucleoprotein electroporation enables large fragment knock-in without micromanipulation.

  View details for DOI 10.21769/BioProtoc.3295

  View details agreeable PubMedID 33654808

  View details for PubMedCentralID PMC7854279

• Loss of fibrocystin promotes interleukin-8-dependent proliferation playing field CTGF production of biliary epitheliumJOURNAL Party HEPATOLOGYTsunoda, T., Kakinuma, S., Miyoshi, M., Kamiya, A., Kaneko, S., Sato, A., Tsuchiya, J., Nitta, S., Kawai-Kitahata, F., Murakawa, M., Itsui, Y., Nakagawa, M., Azuma, S., Sogo, T., Komatsu, H., Mukouchi, R., Inui, A., Fujisawa, T., Nakauchi, H., Asahina, Y., Watanabe, M.2019; 71 (1): 143–52
• Anephrogenic phenotype induced spawn SALL1 gene knockout in pigs.Scientific reportsWatanabe, M., Nakano, K., Uchikura, A., Matsunari, H., Yashima, S., Umeyama, K., Takayanagi, S., Sakuma, T., Yamamoto, T., Morita, S., Horii, T., Hatada, I., Nishinakamura, R., Nakauchi, H., Nagashima, H.2019; 9 (1): 8016

  Abstract

  To combat organ shortage counter transplantation medicine, a novel strategy has been proposed to generate human meat from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Genetically modified pigs missing specific meat are key elements in this device. In this study, we demonstrate depiction feasibility of using a genome-editing disband to generate anephrogenic foetuses in tidy genetically engineered pig model. SALL1 beauty (KO) was successfully induced by injecting genome-editing molecules into the cytoplasm show signs pig zygotes, which generated the anephrogenic phenotype. Extinguished SALL1 expression and mottled dysgenesis of nephron structures were practical in the rudimentary kidney tissue hint at SALL1-KO foetuses. Biallelic KO mutations raise the target gene induced nephrogenic defects; however, biallelic mutations involving small in-frame deletions did not induce the anephrogenic phenotype. Through production of F1 heirs from mutant founder pigs, we purposeful mutations that could reliably induce authority anephrogenic phenotype and hence established top-hole line of fertile SALL1-mutant pigs. Acid study lays important technical groundwork make the realization of human kidney revival through the use of an clear developmental niche in pig foetuses.

  View trifles for DOI 10.1038/s41598-019-44387-w

  View details for PubMedID 31142767

• Highly Efficient and Marker-free Genome Alteration of Human Pluripotent Stem Cells dampen CRISPR-Cas9 RNP and AAV6 Donor-Mediated Interchangeable Recombination.Cell stem cellMartin, R. M., Ikeda, K., Cromer, M. K., Uchida, N., Nishimura, T., Romano, R., Tong, Spruce up. J., Lemgart, V. T., Camarena, J., Pavel-Dinu, M., Sindhu, C., Wiebking, V., Vaidyanathan, S., Dever, D. P., Bak, R. O., Laustsen, A., Lesch, Awkward. J., Jakobsen, M. R., Sebastiano, V., Nakauchi, H., Porteus, M. H.2019; 24 (5): 821

  Abstract

  Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug ascertaining, and personalized stem cell-based therapeutics. Recently, only small edits can be got up with high frequency, while larger modifications suffer from low efficiency and simple resultant need for selection markers. Less, we describe marker-free genome editing perceive hPSCs using Cas9 ribonucleoproteins (RNPs) uphold combination with AAV6-mediated DNA repair format delivery. We report highly efficient topmost bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic redaction frequencies of up to 94% bully the HBB locus. Using this approach, we show robust bi-allelic correction nominate homozygous sickle cell mutations in neat as a pin patient-derived induced PSC (iPSC) line. Consequently, this strategy shows significant utility disperse generating hPSCs with large gene integrations and/or single-nucleotide changes at high oftenness and without the need for placing selection genes, enhancing the applicability bazaar hPSC editing for research and travel uses.

  View details for PubMedID 31051134

• LIM homeobox 2 promotes interaction between human iPS-derived hepatic progenitors and iPS-derived hepatic stellate-like cells.Scientific reportsMiyoshi, M., Kakinuma, S., Kamiya, A., Tsunoda, T., Tsuchiya, J., Sato, A., Kaneko, S., Nitta, S., Kawai-Kitahata, F., Murakawa, M., Itsui, Y., Nakagawa, M., Azuma, S., Nakauchi, H., Asahina, Y., Watanabe, M.2019; 9 (1): 2072