Targeted radionuclide therapy against cancer stem cell-specific markers, such as CD133, constitutes a promising strategy to eliminate resilient cancer stem cells for improved outcomes in refractory tumors. Here, we report the synthesis and evaluation of [177Lu]Lu-DOTA-RW03, a CD133-targeted radioimmunotherapy.

When two lymphomas occur concurrently or sequentially in a patient, it is a major question whether they derive from the same lymphocyte or hematopoietic precursor cell or developed independently. We studied four composite classic Hodgkin lymphomas (HL) and other mature B-cell lymphomas, and two composite mature B- and T-cell neoplasias by whole exome sequencing (WES). Analysis of their IGV genes revealed that three composite B-cell lymphomas originated from common germinal center-experienced B cells. WES identified shared somatic mutations in the lymphomas of these clonally related composite lymphomas, indicating their derivation from a common, pre-malignant precursor. Most mutations were restricted to one or the other of these lymphomas, likely explaining how distinct lymphomas developed from a common ancestral B cell. In the two B-cell/T-cell lymphoma cases, and a composite clonally unrelated HL/chronic lymphocytic leukemia, the lymphoma partners did not share any somatic mutations. In three cases, we identified potentially oncogenic variants also in cells serving as constitutional controls. These variants may have contributed to development of a composite lymphoma/leukemia. We provide additional evidence of frequent clonal relation in composite lymphomas, highlight the multistep transformation process of related lymphomas with a likely pre-malignant intermediate common precursor, and support the importance of constitutional variants in lymphomagenesis.

Acute myeloid leukemia (AML) is a common hematopoietic malignancy with high recurrence rates, and there is an urgent need for new therapeutic agents. T-cell immunoglobulin mucin-3 (TIM-3) is expressed on the surface of both LSCs and blasts in most AML patients, but not in normal hematopoietic stem cells (HSCs). We have developed KK2845, an antibody drug conjugate (ADC) that consists of an anti-TIM-3 fully human IgG1 antibody, a valine-alanine linker and a highly potent DNA cross-linking pyrrolobenzodiazepine (PBD) dimer SG3199. KK2845 exhibited potent cytotoxicity against AML cells both in vitro and in vivo. The cytotoxicity against AML cells was almost comparable between KK2845 and CD33-ADC, an anti-CD33 antibody conjugated with PBD dimer that has shown high remission rates in clinical studies. In addition to the cytotoxicity depending on PBD dimer, KK2845 also showed potent antibody-dependent cell cytotoxicity (ADCC) activity against AML cells. KK2845 showed less cytotoxicity against human normal bone marrow cells than CD33-ADC. The pharmacokinetics of KK2845 in cynomolgus monkey after intravenous infusion demonstrated a favorable profile. Taken together, these data suggest that KK2845 could be a novel ADC therapeutic in AML.

The modulation of microglial reactivity has emerged as a potential target for developing ischemic stroke therapies. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) possess immunomodulatory properties that may influence microglial responses following ischemia. However, individual studies assessing this influence have provided limited results. Therefore, we conducted a systematic review and meta-analysis to investigate whether MSC-EVs treatment alters microglial responses in animal and cellular models of ischemic stroke. In accordance with the PRISMA 2020 statement, we searched PubMed, Web of Science, and EMBASE until October 2023 for studies assessing cellular and molecular parameters of microglial reactivity following MSC-EVs treatment in models of ischemic stroke. We estimated treatment effects using a random-effects meta-analysis of standardized mean differences and estimated heterogeneity via the I2 statistic. The risk of bias was assessed using the SYRCLE questionnaire. The search identified 297 studies, 27 of which met the inclusion criteria. In animal models, MSC-EVs reduced the number, surface area, and fluorescence intensity of Iba1+ cells, as well as the number of Iba1+ cells co-expressing the pro-inflammatory markers CD16, CD32, CD85, and iNOS. Conversely, MSC-EVs increased the number of Iba1+ cells co-expressing the anti-inflammatory markers Arg-1 and CD206. In cellular models, we observed decreased concentrations of TNF-α, IL-1β, and IL-6 in the culture medium. Our meta-analysis consolidates the immunomodulatory effects of MSC-EVs on microglial responses to ischemia, underscoring the potential of microglia-specific therapeutics in the development of MSC-EVs-based and regenerative treatments for ischemic stroke.

The precise location and functions of N6-methyladenosine (m6A) modification on mammalian nuclear noncoding RNA remain largely unknown. Here we developed nuclear-m6A-label-seq to directly map human and mouse cell nuclear RNA m6A methylome at single-base resolution. Specifically, m6A modifications have been identified on abundant human γ satellite DNA II (GSATII) RNA transcripts, a type of repeat RNA, transcribed from SST1-TAR1-GSATII satellite arrays in the pericentromeric region of chromosome 9. GSATII RNA m6A positively regulates the transcription of GSATII-located satellite arrays as well as trans-associated peri/centromeric satellites, typically chromosome 3 centromeric higher-order repeat α satellite. Dysregulation of this circuit renders a phenotype of abnormal chromosome segregation. Mechanistic study reveals that YTHDC1 reads GSATII RNA m6A marks and recruits bromodomain protein 4 (BRD4) to promote transcriptions of the associated satellites via an m6A-YTHDC1-BRD4-H3K27ac axis. These results uncover a mechanism governing the transcription of cis- and trans-associated pericentromeric and centromeric satellites via cross-talk between epitranscriptomic and epigenomic marks.

Bone defects are common orthopedic conditions, and due to the limited regenerative capacity of bone tissue, their repair remains a challenge in orthopedic surgery. Mesenchymal stem cells (MSCs) have demonstrated strong potential for osteogenic differentiation; however, their efficiency in vivo remains restricted, particularly in terms of differentiation and migration. Photobiomodulation (PBM), a non-invasive therapeutic technique, has shown great promise in promoting stem cell differentiation. In this study, we cultured human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro and treated them with 635/808 nm laser light. We measured alkaline phosphatase (ALP) activity, mineralized nodule formation, and the expression of osteogenesis-related genes and factors after 7, 14, and 21 days. The results showed that PBM treatment significantly enhanced hUCMSC proliferation and osteogenic differentiation. The mechanism behind this effect involves PBM activating the upstream Akt signaling pathway, increasing P-Akt expression, and elevating reactive oxygen species (ROS) levels to induce mild oxidative stress. This process enhances ALP activity, mineralized nodule formation, and the expression of osteogenesis-related genes and factors, thus promoting the osteogenic differentiation of hUCMSCs.

DNA polymerase γ (POLγ), responsible for mitochondrial DNA replication, consists of a catalytic POLγA subunit and two accessory POLγB subunits. Mutations in POLG, which encodes POLγA, lead to various mitochondrial diseases. We investigated the most common POLG mutations (A467T, W748S, G848S, Y955C) by characterizing human and mouse POLγ variants. Our data reveal that these mutations significantly impair POLγ activities, with mouse variants exhibiting milder defects. Cryogenic electron microscopy highlighted structural differences between human and mouse POLγ, particularly in the POLγB subunit, which may explain the higher activity of mouse POLγ and the reduced severity of mutations in mice. We further generated a panel of mouse models mirroring common human POLG mutations, providing crucial insights into the pathogenesis of POLG-related disorders and establishing robust models for therapeutic development. Our findings emphasize the importance of POLγB in modulating the severity of POLG mutations.

Stem cell-derived islet cell therapy can effectively treat type 1 diabetes, but its efficacy is hindered by low oxygen supply post-transplantation, particularly in subcutaneous spaces and encapsulation devices, leading to cell dysfunction. The response to hypoxia and effective strategies to alleviate its detrimental effects remain poorly understood. Here, we show that β cells within stem cell-derived islets gradually undergo a decline in cell identity and metabolic function in hypoxia. This is linked to reduced expression of immediate early genes (EGR1, FOS, and JUN), which downregulates key β cell transcription factors. We further identified genes important for maintaining β cell fitness in hypoxia, with EDN3 as a potent player. Elevated EDN3 expression preserves β cell identity and function in hypoxia by modulating genes involved in β cell maturation, glucose sensing and regulation. These insights improve the understanding of hypoxia's impact on stem cell-derived islets, offering a potential intervention for clinical applications.

Sodium benzoate (NaB) is a commonly used food ingredient that is also found in cosmetics and medicines. Previous studies have demonstrated that long-term NaB intake has detrimental effects on human health, while its effects on bone mass remain unknown. In the present study, intragastric NaB administration was found to decrease bone mass and deteriorate bone microstructure in vivo, while prolonged NaB gavage further accelerated bone loss. The in vitro study revealed that NaB inhibited osteoblast differentiation of bone marrow mesenchymal stem cells and MC3T3-E1 cells. Mechanistically, RNA sequencing analysis elucidated that NaB greatly suppressed fibroblast growth factor 2 (FGF2) expression. Further studies revealed that NaB inhibited p38/RUNX2 signaling transduction, which was downstream of FGF2 for modulating osteoblast differentiation. The rescue studies suggested that NaB inhibited RUNX2 expression and osteoblast differentiation through the p38/MAPK signaling pathway. Collectively, NaB accelerated bone loss by inhibiting osteoblast differentiation through downregulating FGF2/p38/RUNX2 signaling pathway. The present study revealed that the long-term intake of NaB-containing food increased the risk of bone loss and osteoporosis (OP). Therefore, a reasonable oral intake of NaB-containing food is an important but convenient initiative for preventing OP.

The remarkable cell diversity of multicellular organisms relies on the ability of multipotent progenitor cells to generate distinct cell types at the right times and locations during embryogenesis. A key question is how progenitors establish competence to respond to the different environmental signals required to produce specific cell types at critical developmental time points. We addressed this in the mouse developing forebrain, where neural progenitor cells must switch from producing neurons to making oligodendrocytes in response to increased Sonic hedgehog (SHH) signaling during late embryogenesis. We show that progenitor responses to SHH are regulated by Notch signaling, thus permitting proper timing of the neuron-oligodendrocyte switch. Notch activity epigenetically primes genes associated with the oligodendrocyte lineage and SHH pathway, enabling amplified transcriptional responses to endogenous SHH and robust oligodendrogenesis. These results reveal a critical role for Notch in facilitating progenitor competence states and influencing cell fate transitions at the epigenetic level.

Friedreich ataxia (FA) is an autosomal recessive disease characterized by progressive damage to the nervous system and severe cardiac abnormalities. The disease is caused by a GAA•TTC triplet repeat expansion in the first intron of the FXN gene, which results in epigenetic repression of FXN transcription and reduction in FXN (frataxin) protein which results in mitochondrial dysfunction. Factors and pathways that promote FXN repression represent potential therapeutic targets whose inhibition would restore FXN transcription and frataxin protein levels. Here, we performed a candidate-based RNAi screen to identify kinases, a highly druggable class of proteins, that when knocked down upregulate FXN expression. Using this approach, we identified Rho kinase ROCK1 as a critical factor required for FXN repression. ShRNA-mediated knockdown of ROCK1, or the related kinase ROCK2, increases FXN mRNA and frataxin protein levels in FA patient-derived induced pluripotent stem cells (iPSCs) and differentiated neurons and cardiomyocytes to levels observed in normal cells. We demonstrate that small molecule ROCK inhibitors, including the FDA-approved drug belumosudil and fasudil, reactivate FXN expression in cultured FA iPSCs, neurons, cardiomyocytes, and FA patient primary fibroblasts, and ameliorate the characteristic mitochondrial defects in these cell types. Remarkably, treatment of transgenic FA mice of both sexes with belumosudil or fasudil upregulates FXN expression, ameliorates the mitochondrial defects in the brain and heart tissues, and improves motor coordination and muscle strength. Collectively, our study identifies ROCK kinases as critical repressors of FXN expression and provides preclinical evidence that FDA approved ROCK inhibitors may be repurposed for treatment of FA.Significance Statement Friedreich ataxia is a debilitating disorder caused by epigenetic repression of the frataxin (FXN) gene, leading to neurodegeneration and cardiomyopathy. Through an RNA interference screen, we identified ROCK1 and ROCK2 kinases as critical repressors of FXN expression, making them promising therapeutic targets for upregulating FXN in patient-derived cells. Treatment with small-molecule ROCK inhibitors, including the FDA-approved drug belumosudil and clinically advanced fasudil, restores frataxin levels, alleviates mitochondrial defects, and improves disease phenotypes in cells and animal models. These findings establish ROCK kinases as targets for Friedreich ataxia therapy and open new avenues for repurposing existing ROCK inhibitors, warranting clinical exploration.

While the World Marrow Donor Association global database currently offers approximately 42.7 million potential donors and cord blood units to patients in need of haematopoietic cell transplant, lack of eight HLA-matched donors remains a significant barrier. The Registry of Unmet Need (RUN) Project seeks to address disparities in transplant access for patients with rare HLA genotypes, particularly those from populations that have been historically underrepresented and underserved by global donor registries. Patients eligible for this study searched for an unrelated donor for transplant between 2015 and 2017 and, at that time, lacked a potential eight-of-eight HLA-matched unrelated donor (MUD). Sixteen donor registries contributed data from 3654 patients using standardised data-collection project templates. To address this unmet need, pooled data were analysed to identify trends and inform global recruitment strategies. Patient genotypes were queried against the global inventory at later timepoints in 2018 and 2023 to determine whether potential matches had been recruited within the years since the initial search. Patient haplotypes were imputed using an open-source method referencing US population frequencies. The imputation process used five continental reference populations and 21 detailed populations derived from the NMDP database. The method provided a Bayesian inference of population membership. A control group consisting of US patients that yielded 1000 or more potential matches was used for comparison. RUN patient haplotype and genotype frequencies were substantially lower compared with controls; both the more frequent and less frequent haplotypes in RUN patients were found to be approximately 100 times less common than those in the control group. We identified 782 potential cases in which a potential MUD was recruited after the initial RUN patient search was performed; while this result is being further investigated, clear patterns of where these new matches can be found have emerged; typically, new matches are found outside the country where the patient search was initiated. Our findings demonstrate that rare haplotypes are the primary barrier to identifying a MUD; the presence of rare alleles or haplotype combinations, as with multi-race ancestry, is rarely the cause. Although strategic donor recruitment efforts will help improve MUD access, patient transplants should not be delayed in pursuit of a MUD when viable alternative options are available.

Cancer immunotherapeutic CpG oligodeoxynucleotide (ODN) is an agonist for TLR9 and a potent inducer of inflammatory cytokines and type I interferon. Clinical trials of CpG ODNs highlight the urgent need for effective TLR9 agonist CpG ODNs in humans. Here, we developed a highly potent CpG ODN, A602, which induces antitumor immune responses in combination with CXCL14. A602 induced secretion of interferon-α by human peripheral blood mononuclear cells. In mouse macrophages, dendritic cells, and human plasmacytoid dendritic cell lines, CXCL14 enhanced cellular uptake of A602, thereby promoting TLR9-mediated immune responses. Importantly, A602 exhibited strong antitumor activity in syngeneic mouse models of colorectal cancer-derived CT26, B lymphoma-derived A20, and melanoma-derived B16F10 cells. Because the antitumor effect of A602 against B16F10 cells was negated in Cxcl14 knockout mice, endogenously expressed CXCL14 is required for the A602-mediated tumor suppression. Thus, modulation of CXCL14 during the A602-induced immune responses shall unveil an innovative new approach for the antitumor immune therapy.

Dysfunctional endothelium contributes to the initiation and progression of atherosclerosis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates the expression of antioxidant and cytoprotective genes. Reduced activity of Nrf2 can contribute to endothelial dysfunction. While the role of endothelial Nrf2 in vascular homeostasis is well supported, several knowledge gaps remain, particularly regarding its cell type-specific contributions and crosstalk mechanisms in vascular disease progression.

The persistently low success rate of human in vitro fertilization (IVF) remains a major clinical challenge, despite significant technological advancements. While culture media composition is known to influence outcomes, the specific components affecting embryonic development are poorly understood. In this study, we identify N-glycolylneuraminic acid (Neu5Gc), a foreign sialic acid contaminant prevalent in animal-derived IVF media components, as a micro-environmental factor triggering developmental arrest. We demonstrate that Neu5Gc accumulates in arrested human embryos via endocytosis and upregulates p53, leading to caspase-3/PARP -dependent apoptotic pathways. This mechanism was validated in human embryonic stem cells (hESCs) and zebrafish embryos, with antibody-mediated Neu5Gc neutralization rescuing developmental defects. Our findings not only elucidate a novel pathway contributing to IVF inefficiency but also provide actionable insights for optimizing culture media formulations.

Diabetic foot ulcer (DFU), a severe complication of diabetes mellitus, presents significant clinical challenges due to its rapid deterioration and high morbidity rates. While conventional therapies exist kinds of limitations, their clinical utility is frequently constrained. Recent advancements in biomedical engineering have introduced innovative therapeutic modalities, particularly nanomaterials and hydrogels. However, emerging technologies face translational barriers including immature manufacturing processes leading to elevated costs, and insufficient long-term safety data due to limited clinical validation periods. Current clinical studies remain constrained by small cohort sizes and preliminary-stage investigations. The purpose of this study was to review traditional primary treatment and simultaneously combine clinical data to increase the speed of innovative safety, cost, and effectiveness indicator testing.

This study explored the molecular mechanism of adipose-derived stem cell-derived extracellular vesicles (ADSC-EVs) improving post-sepsis-associated acute kidney injury (S-AKI) tubular epithelial cell (TEC) apoptosis by modulating ADAM17/MerTK-mediated macrophage efferocytosis.

Chemotherapy is a cornerstone treatment for acute leukemia (AL), but it often results in bone marrow (BM) failure, leading to infections, anemia, and bleeding, which significantly impact patient survival. Endothelial progenitor cells (EPCs) are critical elements of the BM microenvironment and are essential for hematopoiesis. Our previous research using in vitro and AML mouse models indicated that BM EPC dysfunction, characterized by impaired angiogenesis and elevated reactive oxygen species (ROS) levels in AML patients, could be partially reversed after complete remission (CR) and further improved with N-acetyl-L-cysteine (NAC) treatment. This pilot cohort study (NCT06024031, www.clinicaltrials.gov) evaluated the effects of NAC on hematopoietic recovery in 30 newly diagnosed AML patients after induction chemotherapy, compared to a propensity-matched control group of 60 patients. Patients received oral NAC (400 mg, three times daily) for 28 days post-chemotherapy alongside standard supportive care. NAC treatment did not affect CR rates (90% vs. 80%, P=0.23), but significantly shortened platelet recovery time (19 vs. 22 days, P=0.0001) among CR patients. NAC improved EPC percentages, reduced ROS, and enhanced EPC hematopoiesis-supporting functions in patients who achieved CR. NAC was safe and effective in promoting normal hematopoiesis recovery in AML patients in CR following chemotherapy.

Nonsense-mediated mRNA decay (NMD) eliminates transcripts containing premature termination codons (PTCs), thereby preventing errors in protein synthesis. Serine/Threonine-protein kinase SMG1 is a crucial kinase for NMD response, interacting with other regulatory proteins such as SMG8 and SMG9. We identified a de novo heterozygous variant in SMG1 p.Gln2398Glu (c.7192C>G) in a patient with global developmental delay, facial dysmorphism, and oculomotor apraxia. Thus, stem cell models with SMG1 mutations using gene editing technology were established to address the functional consequences of this mutation. While mutations causing the reduction in SMG1 gene dosage by alterations in splicing (c.7192_7194delinsGAA; GAA/+) or frameshift (c.4331_4337del; KO/+) led to a mild but significant reduction of NMD activity, NMD activity was not altered in cells with the SMG1 GAG/+ mutation. Furthermore, cortical organoids from hPSCGAA/+ exhibited size reduction compared to the control (CTL) or GAG/+, suggesting that reduced NMD activity can affect nervous system development. These findings suggest that hypomorphic SMG1 mutations can cause reduced NMD activity and subsequent biological responses, while the mutation found in the patient alone may not be sufficient to induce pathological symptoms.