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Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here,we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging. View Publication

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However,HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C,in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29,CD73,CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2),OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also,the cells showed high expression of MSC markers CD29,CD73,CD44 and CD105,but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion,HUCB is a good source of MSC using this new technique. View Publication

Cell-autonomous barriers to reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remain poorly understood. Using a focused CRISPR-Cas9 screen,we identified Ubiquitin-specific peptidase 22 (USP22) as a key chromatin-based barrier to human iPSC derivation. Suppression of USP22 significantly enhances reprogramming efficiency. Surprisingly,this effect is likely to be independent of USP22’s deubiquitinase activity or its association with the SAGA complex,as shown through module-specific knockouts,and genetic rescue experiments. USP22 is not required for iPSC derivation or maintenance. Mechanistically,USP22 loss during reprogramming downregulates fibroblast-specific genes while activating pluripotency-associated genes,including DNMT3L,LIN28A,SOX2,and GDF3. Additionally,USP22 loss enhances reprogramming efficiency under naïve stem cell conditions. These findings reveal an unrecognized role for USP22 in maintaining somatic cell identity and repressing pluripotency genes,highlighting its potential as a target to improve reprogramming efficiency. Ubiquitin-specific peptidase 22 (USP22) is identified as a key chromatin-based barrier to human iPSC derivation through a chromatin-focused CRISPR-Cas9 screen. View Publication

Understanding the multicellular organization of stem cells is vital for determining the mechanisms that coordinate cell fate decision-making during differentiation; these mechanisms range from neighbor-to-neighbor communication to tissue-level biochemical gradients. Current methods for quantifying multicellular patterning tend to capture the spatial properties of cell colonies at a fixed scale and typically rely on human annotation. We present a computational pipeline that utilizes topological data analysis to generate quantitative,multiscale descriptors which capture the shape of data extracted from 2D multichannel microscopy images. By applying our pipeline to certain stem cell colonies,we detected subtle differences in patterning that reflect distinct spatial organization associated with loss of pluripotency. These results yield insight into putative directed cellular organization and morphogen-mediated,neighbor-to-neighbor signaling. Because of its broad applicability to immunofluorescence microscopy images,our pipeline is well-positioned to serve as a general-purpose tool for the quantitative study of multicellular pattern formation. View Publication

Spaceflight and microgravity environments have been shown to cause significant health impairments,including bone loss,immune dysfunction,and hematopoietic disorders. Hematopoietic stem cells (HSCs),as progenitors of the hematopoietic system,are critical for the continuous renewal and regulation of immune cells. Therefore,elucidating the regulatory mechanisms governing HSC fate and differentiation in microgravity environments is of paramount importance. In this study,hindlimb unloading (HU) was employed in mice to simulate microgravity conditions. After 28 days of HU,cells were isolated for analysis. Flow cytometry and colony-forming assays were utilized to assess changes in HSC proliferation and differentiation. Additionally,transcriptomic and untargeted metabolomic sequencing were performed to elucidate alterations in the metabolic pathways of the bone marrow microenvironment and their molecular regulatory effects on HSCs fate. Our findings revealed that 28 days of HU impaired hematopoietic function,leading to multi-organ damage and hematological disorders. The simulated microgravity environment significantly increased the HSCs population in the bone marrow,particularly within the long-term and short-term subtypes,while severely compromising the differentiation capacity of hematopoietic stem/progenitor cells. Transcriptomic analysis of HSCs,combined with metabolomic profiling of bone marrow supernatants,identified 1,631 differentially expressed genes and 58 metabolites with altered abundance. Gene set enrichment analysis indicated that HU suppressed key pathways,including hematopoietic cell lineage and MAPK signaling. Furthermore,integrated analyses revealed that metabolites affected by HU,particularly hypoxanthine enriched in the purine metabolism pathway,were closely associated with hematopoietic cell lineage and MAPK signaling pathways. Molecular docking simulations and in vitro experiments confirmed that hypoxanthine interacts directly with core molecules within these pathways,influencing their expression. These findings demonstrate that hypoxanthine in the bone marrow supernatant acts as a signaling mediator under microgravity,influencing HSCs fate by modulating hematopoietic cell lineage and MAPK signaling pathways. This study offers novel insights into the impact of microgravity on HSC fate and gene expression,underscoring the pivotal role of bone marrow microenvironmental metabolic changes in regulating key signaling pathways that determine hematopoietic destiny. The online version contains supplementary material available at 10.1186/s13287-025-04213-9. View Publication

Alzheimer’s disease (AD) is characterized by the accumulation and spread of Tau intraneuronal inclusions throughout most of the telencephalon,leaving hindbrain regions like the cerebellum and spinal cord largely spared. These neuropathological observations,along with the identification of specific vulnerable sub-populations from AD brain-derived single nuclei transcriptomics,suggest that a subset of brain regions and neuronal subtypes possess a selective vulnerability to Tau pathology. Given the inability to culture neurons from patient brains,a disease-relevant in vitro model which recapitulates these features would serve as a critical tool to validate modulators of vulnerability and resilience. Using our recently established platform for inducing endogenous Tau aggregation in human induced pluripotent stem cell (hiPSC)-derived cortical excitatory neurons via application of AD brain-derived exogenous Tau aggregates,we explored whether Tau aggregates preferentially induce aggregation in specific neuronal subtypes. We compared Tau seeding in hiPSC-derived neuron subtypes representing regional identities across the forebrain,midbrain,and hindbrain. Higher susceptibility (i.e. more Tau aggregation) was consistently observed among cortical neuron subtypes,with CTIP2-positive,somatostatin (SST)-positive cortical inhibitory neurons showing the greatest aggregation levels across hiPSC lines from multiple donors. hiPSC-neurons also delineated between the disease-specific vulnerabilities of different protein aggregates,as α-synuclein preformed fibrils showed an increased propensity to induce aggregates in midbrain dopaminergic (mDA)-like neurons,mimicking Parkinson’s disease (PD)-specific susceptibility. Aggregate uptake and degradation rates were insufficient to explain differential susceptibility. The absence of a consistent transcriptional response following aggregate seeding further indicated that intrinsic neuronal subtype-specific properties could drive susceptibility. The present data provides evidence that hiPSC-neurons exhibit features of selective neuronal vulnerability which manifest in a cell autonomous manner,suggesting that mining intrinsic (or basal) transcriptomic signatures of more vulnerable compared to more resilient hiPSC-neurons could uncover the molecular underpinnings of differential susceptibility to protein aggregation found in a variety of neurodegenerative diseases. The online version contains supplementary material available at 10.1186/s40478-025-02000-4. View Publication

Cost-effective,practical,and reproducible culture of human pluripotent stem cells (hPSCs) is required for basic and translational research. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hPSC culture. However,the requirement to home-produce some recombinant growth factors for B8 can hinder access and reproducibility. Moreover,we found the published B8 formulation suboptimal in widely-used normoxic hPSC culture. Lastly,the performance of B8 in functional applications such as genome editing or organoid differentiation required systematic evaluation. We formulated B8 with commercially available,growth factors and adjusted its composition to support normoxic culture of WTC11 human induced pluripotent stem cell line. We compared this formulation (B8+) with commercial Essential 8 (cE8) and a home-made,weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle by flow cytometry,and investigated the transcriptional profiles by bulk and single-cell RNA sequencing. We further assessed genomic stability,genome editing efficiency,single-cell cloning,and differentiation in both monolayer and organoids. Finally,we validated key findings using male (H1) and female (H9) human embryonic stem cells. hE8 performed comparably to cE8 across most functional assays and cell lines. In contrast,cells in B8+ displayed higher NANOG expression and improved genome editing efficiency. At the same time,B8+ led to gene expression changes indicative of marked lineage priming,reflected in altered morphology and differential response to some differentiation protocols. Both weekend-free media resulted in a modest transcriptional shift towards a less metabolically active state,consistent with intermittent media starvation. Homemade weekend-free media can provide a cost-effective alternative to commercial formulations. hE8,integrating some features of B8 while resembling cE8,emerges as a robust and practical option with limited compromises. B8+,though advantageous in some contexts,warrants caution due to lineage priming effects that may impact differentiation outcomes. View Publication

Microelectrode material and cell culture medium have significant roles in the signal-to-noise ratio and cell well-being in in vitro electrophysiological studies. Here,we report an ion beam assisted e-beam deposition (IBAD) based process as an alternative titanium nitride (TiN) deposition method for sputtering in the fabrication of state-of-the-art TiN microelectrode arrays (MEAs). The effects of evaporation and nitrogen flow rates were evaluated while developing the IBAD TiN deposition process. Moreover,the produced IBAD TiN microelectrodes were characterized by impedance,charge transfer capacity (CTC) and noise measurements for electrical properties,AFM and SEM for topological imaging,and EDS for material composition. The impedance (at 1 kHz) of brand new 30 $\mu$m IBAD TiN microelectrodes was found to be double but still below 100 k$\Omega$ compared with commercial reference MEAs with sputtered TiN microelectrodes of the same size. On the contrary,the noise level of IBAD TiN MEAs was lower compared with that of commercial sputtered TiN MEAs in equal conditions. In CTC IBAD TiN electrodes (3.3 mC/cm2) also outperformed the sputtered counterparts (2.0 mC/cm2). To verify the suitability of IBAD TiN microelectrodes for cell measurements,human pluripotent stem cell (hPSC)-derived neuronal networks were cultured on IBAD TiN MEAs and commercial sputtered TiN MEAs in two different media: neural differentiation medium (NDM) and BrainPhys (BPH). The effect of cell culture media to hPSC derived neuronal networks was evaluated to gain more stable and more active networks. Higher spontaneous activity levels were measured from the neuronal networks cultured in BPH compared with those in NDM in both MEA types. However,BPH caused more problems in cell survival in long-term cultures by inducing neuronal network retraction and clump formation after 1-2 weeks. In addition,BPH was found to corrode the Si3N4 insulator layer more than NDM medium. The developed IBAD TiN process gives MEA manufacturers more choices to choose which method to use to deposit TiN electrodes and the medium evaluation results remind that not only electrode material but also insulator layer and cell culturing medium have crucial role in successful long term MEA measurements. View Publication

PURPOSE PARP-3 is member of the PARP family of poly (ADP-ribose) polymerases involved in ADPribosylation. PARPs are involved in the basic mechanisms of DNA repair. PARP3,a critical player for efficient mitotic progression,is required for the stabilization of the mitotic spindle by regulation of the mitotic components,NuMA and Tankyrase 1. METHODS The sensitization effect of vinorelbine on PARP3 inhibition-induced cytotoxicity was assessed by the SRB assay. The contribution of programed cell death and cell cycle arrest to the sensitization effect were determined by assessing changes in Annexin V,a marker of apoptosis. Alterations in cell cycle progression were assessed by cell cycle analysis. We used immunofluorescence to assess the effect of vinorelbine and/or PARP3 inhibitors on tubulin and microtubule depolarization. The PARP3 chemiluminescent assay kit was used for PARP3 activity. RESULTS PARP3 inhibitors sensitize breast cancer cells to vinorelbine,a vinca alkaloid used in the treatment of metastatic breast cancer. Olaparib which was originally described as a PARP1 and 2 inhibitor has recently been shown to be a potent PARP3 inhibitor while ME0328 is a more selective PARP3 inhibitor. The combination of vinorelbine with nontoxic concentrations of ME0328 or olaparib reduces vinorelbine resistance by 10 and 17 fold,respectively,potentiating vinorelbine-induced arrest at the G2/M boundary. In addition,PARP3 inhibition potentiates vinorelbine interaction with tubulin. Furthermore,olaparib or ME0328 potentiates vinorelbine-induced PARP3 inhibition,mitotic arrest,and apoptosis. CONCLUSION Our results indicated this approach with PARP3 inhibitors and vinorelbine is unique and promising for breast cancer patients with metastases. This combination could significantly increase the survival of breast cancer patients with metastases. View Publication

Mantle cell lymphoma (MCL) is a clinically aggressive and incurable form of non-Hodgkin lymphoma with very heterogeneous clinical and biological behaviors. Dysregulation of RNA splicing machinery is common in various types of cancer,including hematologic malignancies,and is associated with cancer progression. However,whether and how splicing factors,the spliceosome components responsible for pre-mRNA splicing,regulate MCL aggressive behaviors remain largely unknown. Bioinformatics analyses were employed to profile the mRNA expression of two key families of splicing factors,that are serine/arginine-rich (SR) and heterogenous nuclear ribonucleoprotein (hnRNP) families,in clinical specimens of MCL patients in comparison to normal B cells. Functional roles of splicing factors in MCL aggressive phenotypes defined as hallmarks of cancer were investigated in MCL cell lines. Kaplan–Meier survival analyses were performed to determine the clinical significance of splicing factors according to their gene expression level. Depletion of SRSF1,hnRNP F,and PTBP1,which are highly expressed in MCL clinical specimens,by CRISPR/Cas9 system significantly inhibit cell growth and proliferation,motility,and angiogenesis of MCL cells. SRSF1,hnRNP F (for Z-138 cells),and PTBP1 were found to mediate BTZ sensitivity in MCL cells,in agreement with an increase in caspase-3 activation and the occurrence of splicing events that favor the expression of pro-apoptotic isoforms of apoptosis regulatory genes. We also found that depletion of SRSF1,hnRNP F,and PTBP1 induces the expression of autophagy-related genes and the accumulation of autophagic vacuoles,which may act as one of the tumor-suppressive mechanisms. Survival analyses revealed that co-high expression of SRSF1,HNRNPF,or PTBP1 and oncogenic MYC predicts poor clinical outcomes in MCL patients. Our data describe the clinical significance of aberrant SRSF1,hnRNP F,and PTBP1 in MCL and their tumor-promoting roles via the regulation of cancer hallmarks,which could be important in understanding MCL pathogenesis and therapeutic development. View Publication

During hematopoietic differentiation of human embryonic stem cells (hESCs),early hematopoietic progenitors arise along with endothelial cells within the CD34(+) population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays,their phenotype has not been defined. Here,using hESC differentiation in coculture with OP9 stromal cells,we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45,persisted on differentiating hematopoietic cells,and reliably separated the hematopoietic CD34(+) population from CD34(+)CD43(-)CD31(+)KDR(+) endothelial and CD34(+)CD43(-)CD31(-)KDR(-) mesenchymal cells. Furthermore,we demonstrated that the first-appearing CD34(+)CD43(+)CD235a(+)CD41a(+/-)CD45(-) cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34(+)CD43(+)CD41a(-)CD235a(-)CD45(-) cells. These cells were negative for lineage-specific markers (Lin(-)),expressed KDR,VE-cadherin,and CD105 endothelial proteins,and expressed GATA-2,GATA-3,RUNX1,C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34(+)CD43(+)CD45(-)Lin(-) cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34(+)CD43(+)CD45(+)Lin(-) cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3(high)GATA3(low)RUNX1(low)PU1(high)MPO(high)IL7RA(high) gene expression profile. View Publication

The signals that control the regenerative ability of hematopoietic stem cells (HSCs) in response to damage are unknown. Here,we demonstrate that downstream activation of the Hedgehog (Hh) signaling pathway induces cycling and expansion of primitive bone marrow hematopoietic cells under homeostatic conditions and during acute regeneration. However,this effect is at the expense of HSC function,because continued Hh activation during regeneration represses expression of specific cell cycle regulators,leading to HSC exhaustion. In vivo treatment with an inhibitor of the Hh pathway rescues these transcriptional and functional defects in HSCs. Our study establishes Hh signaling as a regulator of the HSC cell cycle machinery that balances hematopoietic homeostasis and regeneration in vivo. View Publication