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Due to their broad differentiation potential,pluripotent stem cells (PSCs) offer a promising approach for generating relevant cellular models for various applications. While human PSC-based cellular models are already advanced,similar systems for non-human primates (NHPs) are still lacking. However,as NHPs are the most appropriate animals for evaluating the safety of many novel pharmaceuticals,the availability of in vitro systems would be extremely useful to bridge the gap between cellular and animal models. Here,we present a NHP in vitro endothelial cell system using induced pluripotent stem cells (IPSCs) from Cynomolgus monkey (Macaca fascicularis). Based on an adapted protocol for human IPSCs,we directly differentiated macaque IPSCs into endothelial cells under chemically defined conditions. The resulting endothelial cells can be enriched using immuno-magnetic cell sorting and display endothelial marker expression and function. RNA sequencing revealed that the differentiation process closely resembled vasculogenesis. Moreover,we showed that endothelial cells derived from macaque and human IPSCs are highly similar with respect to gene expression patterns and key endothelial functions,such as inflammatory responses. These data demonstrate the power of IPSC differentiation technology to generate defined cell types for use as translational in vitro models to compare cell type-specific responses across species. View Publication

FBW7 is a crucial component of an SCF-type E3 ubiquitin ligase,which mediates degradation of an array of different target proteins. The Fbw7 locus comprises three different isoforms,each with its own promoter and each suspected to have a distinct set of substrates. Most FBW7 targets have important functions in developmental processes and oncogenesis,including Notch proteins,which are functionally important substrates of SCF(Fbw7). Notch signalling controls a plethora of cell differentiation decisions in a wide range of species. A prominent role of this signalling pathway is that of mediating lateral inhibition,a process where exchange of signals that repress Notch ligand production amplifies initial differences in Notch activation levels between neighbouring cells,resulting in unequal cell differentiation decisions. Here we show that the downstream Notch signalling effector HES5 directly represses transcription of the E3 ligase Fbw7β,thereby directly bearing on the process of lateral inhibition. Fbw7(Δ/+) heterozygous mice showed haploinsufficiency for Notch degradation causing impaired intestinal progenitor cell and neural stem cell differentiation. Notably,concomitant inactivation of Hes5 rescued both phenotypes and restored normal stem cell differentiation potential. In silico modelling suggests that the NICD/HES5/FBW7β positive feedback loop underlies Fbw7 haploinsufficiency. Thus repression of Fbw7β transcription by Notch signalling is an essential mechanism that is coupled to and required for the correct specification of cell fates induced by lateral inhibition. View Publication

Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7,P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture,but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive,glucose-transporter-2-low (Ins(+)Glut2(LO)) cells,representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7,were retained into adulthood,and a subset differentiated into endocrine,ductal,and neural lineages,illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new,functional β-cells,and which may be potentially exploited for regenerative therapies in the future. View Publication

BackgroundGenome editing in human iPS cells is a powerful approach in regenerative medicine. CRISPR-Cas9 is the most common genome editing tool,but it often induces byproduct insertions and deletions in addition to the desired edits. Therefore,genome editing of iPS cells produces diverse genotypes. Existing assays mostly analyze genome editing results in cell populations,but not in single cells. However,systematic profiling of genome editing outcomes in single iPS cells was lacking. Due to the high mortality of human iPS cells as isolated single cells,it has been difficult to analyze genome-edited iPS cell clones in a high-throughput manner.MethodsIn this study,we developed a method for high-throughput iPS cell clone isolation based on the precise robotic picking of cell clumps derived from single cells grown in extracellular matrices. We first introduced point mutations into human iPS cell pools by CRISPR-Cas9. These genome-edited human iPS cells were dissociated and cultured as single cells in extracellular matrices to form cell clumps,which were then isolated using a cell-handling robot to establish genome-edited human iPS cell clones. Genome editing outcomes in these clones were analyzed by amplicon sequencing to determine the genotypes of individual iPS cell clones. We identified and distinguished the sequences of different insertions and deletions induced by CRISPR-Cas9 while determining their genotypes. We also cryopreserved the established iPS cell clones and recovered them after determining their genotypes.ResultsWe analyzed over 1,000 genome-edited iPS cell clones and found that homozygous editing was much more frequent than heterozygous editing. We also observed frequent homozygous induction of identical genetic manipulations,including insertions and deletions,such as 1-bp insertions and 8-bp deletions. Moreover,we successfully cryopreserved and then recovered genome-edited iPS cell clones,demonstrating that our cell-handling robot-based method is valuable in establishing genome-edited iPS cell clones.ConclusionsThis study revealed a previously unknown property of genome editing in human iPS cells that identical sequence manipulations tend to be induced in both copies of the target sequence in individual cells. Our new cloning method and findings will facilitate the application of genome editing to human iPS cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04414-2. View Publication

Myocardial infarction (MI) is a major global health concern. Although myeloid cells are crucial for tissue repair in emergency haematopoiesis after MI,excessive myelopoiesis can exacerbate scarring and impair cardiac function. Bone marrow (BM) haematopoietic stem cells (HSCs) have the unique capability to replenish the haematopoietic system,but their role in emergency haematopoiesis after MI has not yet been established. Here we collected human sternal BM samples from over 150 cardiac surgery patients,selecting 49 with preserved cardiac function. We show that MI causes detrimental transcriptional and functional changes in human BM HSCs. Lineage tracing experiments suggest that HSCs are contributors of pro-inflammatory myeloid cells infiltrating cardiac tissue after MI. Therapeutically,enforcing HSC quiescence with the vitamin A metabolite 4-oxo-retinoic acid dampens inflammatory myelopoiesis,thereby modulating tissue remodelling and preserving long-term cardiac function after MI. Rettkowski,Romero-Mulero et al. show that myocardial infarction impacts bone marrow haematopoietic stem cells and leads to inflammatory myelopoiesis,which can be dampened by treatment with 4-oxo-retinoic acid,promoting cardiac recovery. View Publication

Strategies targeting leukemic stem and progenitor cells (LSPCs) are needed for durable remissions in acute myeloid leukemia (AML) and high-risk myelodysplastic neoplasms (MDS). While CD123 constitutes a promising target on LSPCs and leukemic blasts,previous CD123-targeting approaches showed limited efficacy and challenging safety profiles. Here,we describe the preclinical efficacy and safety of the bispecific CD123/CD16A innate cell engager “AFM28”,demonstrating superior activity against AML and MDS patient-derived LSPCs and blasts in vitro compared to an Fc-enhanced CD123-targeting antibody,especially towards CD123 low and/or CD64 + leukemic cells. AFM28 induces autologous anti-leukemic activity in fresh AML whole blood cultures,demonstrating its potential to enhance NK cell function from AML patients. Responsiveness can be further enhanced by allogeneic NK cell addition. Anti-leukemic activity of AFM28 is confirmed in xenograft mouse models. In addition,AFM28 is well tolerated and demonstrates pharmacodynamic activity in cynomolgus monkeys. Altogether,our results indicate that AFM28 has the potential to reduce relapse-inducing residual disease and promote long-term remissions for patients with AML and MDS with a favorable safety profile. Subject terms: Cancer immunotherapy,Preclinical research,Acute myeloid leukaemia,Myelodysplastic syndrome View Publication

Activation-induced cytidine deaminase (AID) mediates immunoglobulin class switch DNA recombination (CSR) and somatic hypermutation (SHM),critical processes for maturation of the antibody response. Epigenetic factors,such as histone deacetylases (HDACs),would underpin B cell differentiation stage-specific AID expression. Here,we showed that NAD+-dependent class III HDAC sirtuin 1 (Sirt1) is highly expressed in resting B cells and down-regulated by stimuli inducing AID. B cell Sirt1 down-regulation,deprivation of NAD+ cofactor,or genetic Sirt1 deletion reduced deacetylation of Aicda promoter histones,Dnmt1,and nuclear factor-$\kappa$B (NF-$\kappa$B) p65 and increased AID expression. This promoted class-switched and hypermutated T-dependent and T-independent antibody responses or led to generation of autoantibodies. Genetic Sirt1 overexpression,Sirt1 boost by NAD+,or allosteric Sirt1 enhancement by SRT1720 repressed AID expression and CSR/SHM. By deacetylating histone and nonhistone proteins (Dnmt1 and NF-$\kappa$B p65),Sirt1 transduces metabolic cues into epigenetic changes to play an important B cell-intrinsic role in modulating antibody and autoantibody responses. View Publication

AIMS Contradictory outcomes from recent clinical trials investigating the transplantation of autologous bone marrow mononuclear cell (BM-MNC) fraction containing stem/progenitor cells to damaged myocardium,following acute myocardial infarction,may be,in part,due to the different cell isolation protocols used. We compared total BM-MNC numbers and its cellular subsets obtained following isolation using Ficoll-Paque and Lymphoprep - two different density gradient media used in the clinical trials. MATERIALS & METHODS Bone marrow samples were taken from patients entered into the REGENERATE-IHD clinical trial after 5 days of subcutaneous granulocyte colony-stimulating factor injections. Each sample was divided equally for BM-MNC isolation using Ficoll-Paque and Lymphoprep,keeping all other procedural steps constant. Isolated fractions were characterized for hematopoietic stem cells,endothelial progenitor cells,T lymphocytes,B lymphocytes and NK cells using cell surface markers CD34(+),CD133(+)VEGFR2(+),CD45(+)CD3(+),CD45(+)CD19(+) and CD45(+)CD16(+)CD56(+),respectively. There were no significant differences in the absolute numbers and percentage cell recovery of various mononuclear cell types recovered following separation using either density gradient media. Cell viability and the proportion of various cell phenotypes investigated were similar between the two media. They were also equally efficient in excluding unwanted red blood cells,granulocytes and platelets from the final cell products. CONCLUSION We demonstrated that the composition and quantity of cell types found within therapeutic BM-MNC preparations for use in clinical trials of cardiac stem cell transplantation are not influenced by the type of density gradient media used when comparing Ficoll-Paque and Lymphoprep. View Publication

Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma-null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34(+) human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks),human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses,as documented by the presence of CD4(+) CD8(+) T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation,human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses,the chimerism was weak and the human hematopoietic lineage development was frequently incomplete. View Publication

The increased application of in vitro systems in pharmacology and toxicology requires cell culture systems that facilitate the cultivation process and ensure stable,reproducible and controllable cultivation conditions. Up to now,some devices have been developed for the cultivation of cells under submersed conditions. However,systems meeting the requirements of an air-liquid interface (ALI) cultivation for the special needs of bronchial epithelial cells for example are still lacking. In order to obtain in vivo like organization and differentiation of these cells they need to be cultivated under ALI conditions on microporous membranes in direct contact with the environmental atmosphere. For this purpose,a Long-Term-Cultivation system was developed (CULTEX(®) LTC-C system) for the computer-controlled cultivation of such cells. The transwell inserts are placed in an incubator module (24 inserts),which can be adjusted for the medium level (ultrasonic pulse-echosensor),time and volume-dependent medium exchange,and frequency for mixing the medium with a rotating disc for homogeneous distribution of medium and secretion components. Normal primary freshly isolated bronchial epithelial cells were cultivated for up to 38 days to show the efficiency of such a cultivation procedure for generating 3D cultures exhibiting in vivo-like pseudostratified organization of the cells as well as differentiation characteristics like mucus-producing and cilia-forming cells. View Publication

Human embryonic stem cells (hESCs) are a promising cell source for tissue engineering and regenerative medicine,but before they can be used in therapies,we must be able to accurately identify the state and progeny of hESCs. One of the most commonly used methods for identification is flow cytometry. Many flow cytometry applications use antibodies to detect the amount of antigen present on/in a cell. This allows for the identification of unique cell populations or the tracking of expression changes within a population during differentiation. The results are typically presented as a percentage of positively expressing cells (%Pos) for a marker of choice,relative to a negative control. However,this reporting term is vulnerable to distortion from outliers and inaccuracy from loss of information about the population's fluorescence intensity. In this article,we describe an alternate strategy that uses the normalized median fluorescence intensity (nMFI),in which the MFI of the stained sample is normalized to the MFI of the negative control,as the reporting term to more accurately describe a population of cells in culture. We observed that nMFI provides a more accurate representation for the quality of a starting population and comparing data of different experimental runs. In addition,we demonstrated that the nMFI is a more sensitive measure of pluripotent and differentiation markers expression changes during hESC differentiation into three germ layer lineages. View Publication

Efficient differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to a variety of lineages requires step-wise approaches replicating the key commitment stages found during embryonic development. Here we show that expression of PdgfR-α segregates mouse ESC-derived Flk-1 mesoderm into Flk-1(+)PdgfR-α(+) cardiac and Flk-1(+)PdgfR-α(-) hematopoietic subpopulations. By monitoring Flk-1 and PdgfR-α expression,we found that specification of cardiac mesoderm and cardiomyocytes is determined by remarkably small changes in levels of Activin/Nodal and BMP signaling. Translation to human ESCs and iPSCs revealed that the emergence of cardiac mesoderm could also be monitored by coexpression of KDR and PDGFR-α and that this process was similarly dependent on optimal levels of Activin/Nodal and BMP signaling. Importantly,we found that individual mouse and human pluripotent stem cell lines require optimization of these signaling pathways for efficient cardiac differentiation,illustrating a principle that may well apply in other contexts. View Publication