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Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a mitochondrial disorder characterized by myoclonus epilepsy,generalized seizures,ataxia and myopathy. MERRF syndrome is primarily due to an A to G mutation at mtDNA 8344 that disrupts the mitochondrial gene for tRNA(Lys). However,the detailed mechanism by which this tRNA(Lys) mutation causes mitochondrial dysfunction in cardiomyocytes or neurons remains unclear. In this study,we generated human induced pluripotent stem cells (hiPSCs) that carry the A8344G genetic mutation from patients with MERRF syndrome. Compared with mutation-free isogenic hiPSCs,MERRF-specific hiPSCs (MERRF-hiPSCs) exhibited reduced oxygen consumption,elevated reactive oxygen species (ROS) production,reduced growth,and fragmented mitochondrial morphology. We sought to investigate the induction ability and mitochondrial function of cardiomyocyte-like cells differentiated from MERRF-hiPSCs. Our data demonstrate that that cardiomyocyte-like cells (MERRF-CMs) or neural progenitor cells (MERRF-NPCs) differentiated from MERRF-iPSCs also exhibited increased ROS levels and altered antioxidant gene expression. Furthermore,MERRF-CMs or -NPCs contained fragmented mitochondria,as evidenced by MitoTracker Red staining and transmission electron microscopy. Taken together,these findings showed that MERRF-hiPSCs and MERRF-CM or -NPC harboring the A8344G genetic mutation displayed contained mitochondria with an abnormal ultrastructure,produced increased ROS levels,and expressed upregulated antioxidant genes. View Publication

BACKGROUND Many groups have generated insulin-secreting cells from hESCs/iPSCs in multiple differentiation stages by mimicking the developmental processes. However,these cells do not always secrete glucose responsive insulin,one of the most important characteristics of pancreatic $$ cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic $$ cells. METHODS We established a 6-stage protocol for the differentiation process from hiPSCs to pancreatic $$ cells using defined culture media without feeders or serum. We examined the effect of CHIR99021,the selective inhibitor of GSK-3$$,in the presence of Activin,FGF2,and BMP4 during definitive endodermal induction by immunostaining for SOX17 and FOXA2. We also compared the insulin secretion at the last stage between monolayer culture and spheroid culture conditions. Cultured cells were transplanted under the kidney capsules of STZ-induced diabetic NOD-SCID mice,and blood glucose levels were measured. Immunohistochemical analysis was performed 4 weeks and 12 weeks after transplantation. RESULTS Addition of CHIR99021 in the presence of Activin,FGF2,and BMP4 for 2 days improved the viability of the endodermal cells,keeping the high positive rate of SOX17. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than monolayer culture did. After cell transplantation,diabetic mice showed lowered blood glucose levels,and we detected islet-like structures in vivo. CONCLUSION We generated functional pancreatic $$ cells from human iPS cells. Induction of definitive endoderm and spheroid formation might be key steps for producing them. View Publication

Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds,an activin A signaling inhibitor and a FGF2 signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation,areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC- and placenta-derived syncytial cells are trophoblast,each with the potential to transport a wide range of solutes and synthesize placental hormones,their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall. View Publication

Pluripotent stem cells exhibit cell cycle-regulated heterogeneity for trimethylation of histone-3 on lysine-4 (H3K4me3) on developmental gene promoters containing bivalent epigenetic domains. The heterogeneity of H3K4me3 can be attributed to Cyclin-dependent kinase-2 (CDK2) phosphorylation and activation of the histone methyltransferase,MLL2 (KMT2B),during late-G1. The deposition of H3K4me3 on developmental promoters in late-G1 establishes a permissive chromatin architecture that enables signaling cues to promote differentiation from the G1 phase. These data suggest that the inhibition of MLL2 phosphorylation and activation will prevent the initiation of differentiation. Here,we describe a method to seamlessly modify a putative CDK2 phosphorylation site on MLL2 to restrict its phosphorylation and activation. Specifically,by utilizing dimeric CRISPR RNA-guided nucleases,RFNs (commercially known as the NextGEN™ CRISPR),in combination with an excision-only piggyBac™ transposase,we demonstrate how to generate a point mutation of threonine-542,a predicted site to prevent MLL2 activation. This gene editing method enables the use of both positive and negative selection,and allows for subsequent removal of the donor cassette without leaving behind any unwanted DNA sequences or modifications. This seamless donor-excision" approach provides clear advantages over using single stranded oligo-deoxynucleotides (ssODN) as donors to create point mutations� View Publication

It has been proposed that the ancestral fungus was mating competent and homothallic. However,many mating-competent fungi were initially classified as asexual because their mating capacity was hidden behind layers of regulation. For efficient in vitro mating,the essentially obligate diploid ascomycete pathogen Candida albicans has to change its mating type locus from heterozygous MTL a /α to homozygous MTL a / a or MTL α/α and then undergo an environmentally controlled epigenetic switch to the mating-competent opaque form. These requirements greatly reduce the potential for C. albicans mating. Deletion of the Yci1 domain gene OFR1 bypasses the need for C. albicans cells to change the mating type locus from heterozygous to homozygous prior to switching to the opaque form and mating and allows homothallic mating of MTL heterozygous strains. This bypass is carbon source dependent and does not occur when cells are grown on glucose. Transcriptional profiling of ofr1 mutant cells shows that in addition to regulating cell type and mating circuitry,Ofr1 is needed for proper regulation of histone and chitin biosynthesis gene expression. It appears that OFR1 is a key regulator in C. albicans and functions in part to maintain the cryptic mating phenotype of the pathogen. View Publication

While in vitro liver tissue engineering has been increasingly studied during the last several years,presently engineered liver tissues lack the bile duct system. The lack of bile drainage not only hinders essential digestive functions of the liver,but also leads to accumulation of bile that is toxic to hepatocytes and known to cause liver cirrhosis. Clearly,generation of bile duct tissue is essential for engineering functional and healthy liver. Differentiation of human induced pluripotent stem cells (iPSCs) to bile duct tissue requires long and/or complex culture conditions,and has been inefficient so far. Towards generating a fully functional liver containing biliary system,we have developed defined and controlled conditions for efficient 2D and 3D bile duct epithelial tissue generation. A marker for multipotent liver progenitor in both adult human liver and ductal plate in human fetal liver,EpCAM,is highly expressed in hepatic spheroids generated from human iPSCs. The EpCAM high hepatic spheroids can,not only efficiently generate a monolayer of biliary epithelial cells (cholangiocytes),in a 2D differentiation condition,but also form functional ductal structures in a 3D condition. Importantly,this EpCAM high spheroid based biliary tissue generation is significantly faster than other existing methods and does not require cell sorting. In addition,we show that a knock-in CK7 reporter human iPSC line generated by CRISPR/Cas9 genome editing technology greatly facilitates the analysis of biliary differentiation. This new ductal differentiation method will provide a more efficient method of obtaining bile duct cells and tissues,which may facilitate engineering of complete and functional liver tissue in the future. View Publication

Immune rejection is one of the most serious challenges in allogeneic transplantation,including allogeneic induced pluripotent stem cell (allo-iPSC)-derived cell therapy. Beta-2-Microglobulin gene-knockout,human leukocyte antigen (HLA) class I-deficient iPSCs can evade immune rejection by host T cells,which occurs due to HLA mismatches. However,natural killer (NK) cells recognize HLA class Ⅰ-deficient cells and reject them,which is known as the missing-self response. Introducing chimeric HLA-E protein to HLA class Ⅰ-deficient iPSCs suppresses the missing-self response of NK cells expressing the inhibitory receptor NKG2A; however,technology to suppress NKG2A-negative NK cells is still required. Here,we developed novel agonists for the other inhibitory receptor,killer immunoglobulin receptor (KIR),on NK cells. We found that antibodies that bind to activating KIR enhance NK cell activation and developed selective agonists for inhibitory KIRs (KIR2DL1,KIR2DL2/3,and KIR3DL1). Introducing these selective inhibitory KIR agonists on T cells and HLA class Ⅰ-deficient iPSCs allowed them to evade immune rejection by NK cells. Additionally,we identified an NKG2A-selective agonist as an alternative to chimeric HLA-E,which stimulates the activating receptor NKG2C. This technology enhances immune tolerance in allo-iPSCs and facilitates the development of various iPSC-derived regenerative medicines. The online version contains supplementary material available at 10.1038/s41598-025-18394-z. Subject terms: Allotransplantation,NK cells View Publication

Angelman syndrome is a rare neurodevelopmental disorder caused by the loss of function of the maternally inherited UBE3A gene within the chr15q11-q13 region. This gene is subjected to a tissue-specific form of genomic imprinting leading to the silencing of the paternal allele in neurons. Angelman syndrome can result from various (epi)genetic mechanisms,with paternal uniparental disomy of chromosome 15 (patUPD15) being one of the rarest and least studied due to the absence of suitable models. To address this gap,we generated three independent induced pluripotent stem cell (iPSC) lines from individuals with Angelman syndrome caused by patUPD15,alongside genetically matched unaffected familial controls. Peripheral blood mononuclear cells (PBMCs) were reprogrammed into iPSCs using a non-integrative Sendai virus-based approach expressing the Yamanaka factors. All iPSC lines underwent rigorous quality control,confirming stem cell identity,trilineage differentiation potential,and genetic and epigenetic integrity. This newly established iPSC toolkit provides a powerful platform to investigate the molecular underpinnings of Angelman syndrome caused by patUPD15,paving the way for future translational research and therapeutic development tailored for this understudied form of the disorder. The online version contains supplementary material available at 10.1007/s13577-025-01287-8. View Publication

Autoantibodies against envelope (Env) protein encoded by human endogenous retrovirus group K (HERV-K) are prevalent in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE),but it remains unclear which proviruses are responsible for this autoantigen. It also remains poorly understood how the transcription of HERV-K loci is regulated in cells that can produce Env.ResultsWe aligned our neutrophil RNA sequencing data to the new telomere-to-telomere reference genome and found uniquely mapping transcripts from HERV-K101,K102,K104,K108,K109,K117 and ERVK5,of which only K102,K108,and K109 encode an intact Env. Expression of K102 and K108 were higher in SLE than in healthy donors or RA (padj < 0.05). Transcripts from these proviruses increased in response to interferon-α in monocytes and neutrophils from RA patients and healthy donors,but not in SLE,presumably because they have chronically elevated type I interferons in vivo. Indeed,HERV-K expression was significantly higher in SLE patients with high type I interferon gene signature. Tumor necrosis factor-α and other cytokines and TLR ligands also induced HERV-K102 and K108 transcripts. Interferon-α also increased detectable Env protein in monocytes,macrophages,and neutrophils from RA patients. Among the genes for epigenetic silencers of HERV-K,only TRIM28 was significantly decreased in SLE patients with high interferons (padj = 0.00024).ConclusionsOur data establish a role for interferons in maintaining increased HERV-K expression in SLE and suggest that interferons or other cytokines can upregulate HERV-K to similar levels in RA. A transient increase may also accompany normal immune responses,suggesting that endogenous retroviruses may have been co-opted for efficient immune responses.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13100-025-00371-y. View Publication

Angiotensin (Ang)-(1–7) stimulates vasoprotective functions of diabetic (DB) CD34 + hematopoietic stem/progenitor cells partly by decreasing reactive oxygen species (ROS),increasing nitric oxide (NO) levels and decreasing TGFβ1 secretion. Telomerase reverse transcriptase (TERT) translocates to mitochondria and regulates ROS generation. Alternative splicing of TERT results in variants α-,β- and α-β-TERT,which may oppose functions of full-length (FL) TERT. This study tested if the protective functions of Ang-(1–7) or TGFβ1-silencing are mediated by mitoTERT and that diabetes decreases FL-TERT expression by inducing splicing. CD34 + cells were isolated from the peripheral blood mononuclear cells of nondiabetic (ND,n = 68) or DB (n = 74) subjects. NO and mitoROS levels were evaluated by flow cytometry. TERT splice variants and mitoDNA-lesions were characterized by qPCR. TRAP assay was used for telomerase activity. Decoy peptide was used to block mitochondrial translocation (mitoXTERT). TERT inhibitor or mitoXTERT prevented the effects of Ang-(1–7) on NO or mitoROS levels in DB-CD34 + cells. FL-TERT expression and telomerase activity were lower and mitoDNA-lesions were higher in DB cells compared to ND and were reversed by Ang-(1–7) or TGFβ1-silencing. The prevalence of TERT splice variants,with predominant β-TERT expression,was higher and the expression of FL-TERT was lower in DB cells (n = 25) compared to ND (n = 30). Ang-(1–7) or TGFβ1-silencing decreased TERT-splicing and increased FL-TERT. Blocking of β-splicing increased FL-TERT and protected mitoDNA in DB-cells. The findings suggest that diabetes induces TERT-splicing in CD34 + cells and that β-TERT splice variant largely contributes to the mitoDNA oxidative damage. View Publication

Solid cancers Myeloid cells are prevalent in solid cancers,but they frequently exhibit an anti-inflammatory pro-tumor phenotype that contribute to the immunosuppressive tumor microenvironment (TME),which hinders the effectiveness of cancer immunotherapies. Myeloid cells’ natural ability of tumor trafficking makes engineered myeloid cell therapy an intriguing approach to tackle the challenges posed by solid cancers,including tumor infiltration,tumor cell heterogenicity and the immunosuppressive TME. One such engineering approach is to target the checkpoint molecule PD-L1,which is often upregulated by solid cancers to evade immune responses. Here we devised an adoptive cell therapy strategy based on myeloid cells expressing a Chimeric Antigen Receptor (CAR)-like immune receptor (CARIR). The extracellular domain of CARIR is derived from the natural inhibitory receptor PD-1,while the intracellular domain(s) are derived from CD40 and/or CD3ζ. To assess the efficacy of CARIR-engineered myeloid cells,we conducted proof-of-principle experiments using co-culture and flow cytometry-based phagocytosis assays in vitro. Additionally,we employed a fully immune-competent syngeneic tumor mouse model to evaluate the strategy’s effectiveness in vivo. Co-culturing CARIR-expressing human monocytic THP-1 cells with PD-L1 expressing target cells lead to upregulation of the costimulatory molecule CD86 along with expression of proinflammatory cytokines TNF-1α and IL-1β. Moreover,CARIR expression significantly enhanced phagocytosis of multiple PD-L1 expressing cancer cell lines in vitro. Similar outcomes were observed with CARIR-expressing human primary macrophages. In experiments conducted in syngeneic BALB/c mice bearing 4T1 mammary tumors,infusing murine myeloid cells that express a murine version of CARIR significantly slowed tumor growth and prolonged survival. Taken together,these results demonstrate that adoptive transfer of PD-1 CARIR-engineered myeloid cells represents a promising strategy for treating PD-L1 positive solid cancers. View Publication

Establishing robust models of human myelinating Schwann cells is critical for studying peripheral nerve injury and disease. Stem cell differentiation has emerged as a key human cell model and disease motivating development of Schwann cell differentiation protocols. Human embryonic stem cells (hESCs) are considered the ideal pluripotent cell but ethical concerns regarding their use have propelled the popularity of human induced pluripotent stem cells (hiPSCs). Given that the equivalence of hESCs and hiPSCs remains controversial,we sought to compare the molecular and functional equivalence of hESC- and hiPSC-derived Schwann cells generated with our previously reported protocol. We identified only modest transcriptome differences by RNA sequencing and insignificant proteome differences by antibody array. Additionally,both cell types comparably improved nerve regeneration and function in a chronic denervation and regeneration animal model. Our findings demonstrate that Schwann cells derived from hESCs and hiPSCs with our protocol are molecularly comparable and functionally equivalent. Subject areas: Neuroscience,Cell biology,Stem cells research,Transcriptomics View Publication