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It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study,we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors,by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner,which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors,in a 2-dimensional or 3-dimensional environment. However,Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices,we observed mature Purkinje-like cells with right morphology and marker expression patterns,which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. View Publication

Conventional methods for quantification of undifferentiated pluripotent stem cells such as fluorescence-activated cell sorting and real-time PCR analysis have technical limitations in terms of their sensitivity and recyclability. Herein,we designed a real-time in situ label-free monitoring system on the basis of a specific electrochemical signature of human pluripotent stem cells in vitro. The intensity of the signal of hPSCs highly corresponded to the cell number and remained consistent in a mixed population with differentiated cells. The electrical charge used for monitoring did not markedly affect the proliferation rate or molecular characteristics of differentiated human aortic smooth muscle cells. After YM155 treatment to ablate undifferentiated hPSCs,their specific signal was significantly reduced. This suggests that detection of the specific electrochemical signature of hPSCs would be a valid approach to monitor potential contamination of undifferentiated hPSCs,which can assess the risk of teratoma formation efficiently and economically. View Publication

The aldehyde dehydrogenase (ALDH) superfamily is composed of nicotinamide adenine dinucleotide (phosphate) (NAD(P)(+))-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. To date,24 ALDH gene families have been identified in the eukaryotic genome. In addition to aldehyde metabolizing capacity,ALDHs have additional catalytic (e.g. esterase and reductase) and non-catalytic activities. The latter include functioning as structural elements in the eye (crystallins) and as binding molecules to endobiotics and xenobiotics. Mutations in human ALDH genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases. Most recently ALDH polymorphisms have been associated with gout and osteoporosis. Aldehyde dehydrogenase enzymes also play important roles in embryogenesis and development,neurotransmission,oxidative stress and cancer. This article serves as a comprehensive review of the current state of knowledge regarding the ALDH superfamily and the contribution of ALDHs to various physiological and pathophysiological processes. View Publication

Many long noncoding RNA (lncRNA) species have been identified in mammalian cells,but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (textgreater60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes. View Publication

Induced pluripotent stem cell (iPS cell) holds great potential for applications in regenerative medicine,drug discovery,and disease modeling. We describe here a practical method to generate human iPS cells from urine-derived cells (UCs) under feeder-free,virus-free,serum-free condition and without oncogene c-MYC. We showed that this approach could be applied in a large population with different genetic backgrounds. UCs are easily accessible and exhibit high reprogramming efficiency,offering advantages over other cell types used for the purpose of iPS generation. Using the approach described in this study,we have generated 93 iPS cell lines from 20 donors with diverse genetic backgrounds. The non-viral iPS cell bank with these cell lines provides a valuable resource for iPS cells research,facilitating future applications of human iPS cells. View Publication

BackgroundCHD7 encodes an ATP-dependent chromodomain helicase DNA binding protein; mutations in this gene lead to multiple developmental disorders,including CHARGE (Coloboma,Heart defects,Atresia of the choanae,Retardation of growth and development,Genital hypoplasia,and Ear anomalies) syndrome. How the mutations cause multiple defects remains largely unclear. Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as the thymus,liver,pancreas,and intestine.MethodsIn this study,we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to delete the CHD7 gene in human embryonic stem cells (hESCs) to generate CHD7 homozygous mutant (CHD7?/?),heterozygous mutant (CHD7+/?),and control wild-type (CHD7+/+) cells. We then investigated the ability of the hESCs to develop into DE and the other two germ layers,mesoderm and ectoderm in vitro. We also compared global gene expression and chromatin accessibility among the hESC-DE cells by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with sequencing (ATAC-seq).ResultsWe found that deletion of CHD7 led to reduced capacity to develop into DE and mesoderm in a dose-dependent manner. Loss of CHD7 led to significant changes in the expression and chromatin accessibility of genes associated with several pathways. We identified 40 genes that were highly down-regulated in both the expression and chromatin accessibility in CHD7 deleted hESC-DE cells.ConclusionsCHD7 is critical for DE and mesodermal development from hESCs. Our results provide new insights into the mechanisms by which CHD7 mutations cause multiple congenital anomalies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04437-9. View Publication

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease caused by repeat expansion of the CAG trinucleotide within exon 10 of the ATXN3 gene. This mutation results in the production of an abnormal ataxin-3 protein containing an extended polyglutamine tract,referred to as mutant ataxin-3. In this study,we investigated the therapeutic potential of CRISPR/Cas9-mediated genome editing for SCA3. First,we designed a specific single-guide RNA targeting the ATXN3 gene and constructed the corresponding targeting vector. Induced pluripotent stem cells (iPSCs) derived from a SCA3 patient were then electroporated with the CRISPR/Cas9 components. Positive clones were screened and validated by PCR and Sanger sequencing to obtain genome-editing iPSCs (GE-iPSCs). Subsequently,the pluripotency of GE-iPSCs was confirmed,and the effects of genome editing on mutant ataxin-3 protein expression and Golgi apparatus morphology were assessed using Western blotting and immunofluorescence analyses. Our results demonstrated that targeted insertion of polyadenylation signals (PAS) upstream of the abnormal CAG repeats effectively suppressed the production of mutant ataxin-3. This intervention also reduced the formation of neuronal nuclear inclusions in differentiated neurons,restored the structural integrity of the Golgi apparatus (which exhibited a loose and enlarged morphology in SCA3 cells),and increased the expression levels of Golgi structural proteins (GM130 and GORASP2). In conclusion,our findings indicate that the targeted insertion of PAS upstream of the abnormal CAG repeats in the ATXN3 gene represents a promising therapeutic strategy for SCA3 through genome editing.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-93369-8. View Publication

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However,we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis,particularly in the late stage,where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus,we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge,the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this,we generated inducible TRIB3 overexpression hESCs,named TRIB3tet-on OE H9,based on a Tet-On system. Then,we analyzed hemoglobin expression,condensed chromosomes,organelle clearance,and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry,enhanced hemoglobin and related protein expression in Western blot,decreased nuclear area size,promoted enucleation rate,decreased lysosome and mitochondria number,more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 ?g/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore,our study delineates the role of TRIB3 in terminal erythropoiesis,and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin. Highlights•TRIB3 boosts erythroid cell maturation.•Key insights into erythropoiesis from hESCs.•Enhanced ubiquitin-proteasome system and downstream mitophagy in erythroid differentiation. View Publication

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system,guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study,we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR,immunofluorescence,and Western blot analysis. Additionally,we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes,including CYP450,urea cycle enzymes,and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes,evident as early as 1 day post-differentiation. Interestingly,HLSCs transiently upregulated stem cell markers during differentiation,followed by downregulation after 7 days. Furthermore,differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24 h,with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system,its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications. View Publication

Receptor tyrosine kinases (RTK) are therapeutic targets for the treatment of malignancy. However,tumor cells develop resistance to targeted therapies through the activation of parallel signaling cascades. Recent evidence has shown that redundant or compensatory survival signals responsible for resistance are initiated by nontargeted glycoprotein RTKs coexpressed by the cell. We hypothesized that disrupting specific functions of the posttranslational machinery of the secretory pathway would be an effective strategy to target both primary and redundant RTK signaling. Using the N-linked glycosylation inhibitor,tunicamycin,we show that expression levels of several RTKS (EGFR,ErbB2,ErbB3,and IGF-IR) are exquisitely sensitive to inhibition of N-linked glycosylation. Disrupting this synthetic process reduces both cellular protein levels and receptor activity in tumor cells through retention of the receptors in the endoplasmic reticulum/Golgi compartments. Using U251 glioma and BXPC3 pancreatic adenocarcinoma cell lines,two cell lines resistant to epidermal growth factor receptor-targeted therapies,we show that inhibiting N-linked glycosylation markedly reduces RTK signaling through Akt and radiosensitizes tumor cells. In comparison,experiments in nontransformed cells showed neither a reduction in RTK-dependent signaling nor an enhancement in radiosensitivity,suggesting the potential for a therapeutic ratio between tumors and normal tissues. This study provides evidence that enzymatic steps regulating N-linked glycosylation are novel targets for developing approaches to sensitize tumor cells to cytotoxic therapies. View Publication

SARS-CoV-2,a $\beta$-coronavirus,has rapidly spread across the world,highlighting its high transmissibility,but the underlying morphogenesis and pathogenesis remain poorly understood. Here,we characterize the replication dynamics,cell tropism and morphogenesis of SARS-CoV-2 in organotypic human airway epithelial (HAE) cultures. SARS-CoV-2 replicates efficiently and infects both ciliated and secretory cells in HAE cultures. In comparison,HCoV-NL63 replicates to lower titers and is only detected in ciliated cells. SARS-CoV-2 shows a similar morphogenetic process as other coronaviruses but causes plaque-like cytopathic effects in HAE cultures. Cell fusion,apoptosis,destruction of epithelium integrity,cilium shrinking and beaded changes are observed in the plaque regions. Taken together,our results provide important insights into SARS-CoV-2 cell tropism,replication and morphogenesis. View Publication

Acute Myeloid Leukemia (AML) is caused by multiple mutations which dysregulate growth and differentiation of myeloid cells. Cells adopt different gene regulatory networks specific to individual mutations,maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However,patients harbour a population of quiescent leukemic stem cells (LSCs) which can emerge from quiescence to trigger relapse after therapy. The processes that allow such cells to re-grow remain unknown. Here,we examine the well characterised t(8;21) AML sub-type as a model to address this question. Using four primary AML samples and a novel t(8;21) patient-derived xenograft model,we show that t(8;21) LSCs aberrantly activate the VEGF and IL-5 signalling pathways. Both pathways operate within a regulatory circuit consisting of the driver oncoprotein RUNX1::ETO and an AP-1/GATA2 axis allowing LSCs to re-enter the cell cycle while preserving self-renewal capacity. Subject terms: Cancer stem cells,Acute myeloid leukaemia,Target validation View Publication