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BACKGROUND/AIMS [corrected] Embryonic stem cells (ES cells) have the capacity to propagate indefinitely,maintain pluripotency,and differentiate into any cell type under defined conditions. As a result,they are considered to be the best model system for research into early embryonic development. AICA ribonucleotide (AICAR) is an activator of AMP-activated protein kinase (AMPK) that is thought to affect ES cell function,but its role in ES cell fate decision is unclear. METHODS In this study,we performed microarray analysis to investigate AICAR downstream targets and further understand its effect on ES cells. RESULTS Our microarray data demonstrated that AICAR can significantly up-regulate pluripotency-associated genes and down-regulate differentiation-associated transcription factors. Although AICAR cannot maintain ES cell identity without LIF,it can antagonize the action of RA-induced differentiation. Using those differentially expressed genes identified,we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the Database for Annotation,Visualization and Integrated Discovery (DAVID) online system. AICAR was not only shown to influence the AMPK pathway,but also act on other signaling pathways such as BMP,MAPK and TGF-β,to maintain the stemness of J1 ES cells. Furthermore,AICAR modulated ES cell epigenetic modification by altering the expression of epigenetic-associated proteins,including Dnmt3a,Dnmt3b,Smarca2,Mbd3,and Arid1a,or through regulating the transcription of long intervening non-coding RNA (lincRNA). CONCLUSION Taken together,our work suggests that AICAR is capable of maintaining ES cell self-renewal and pluripotency,which could be useful in future medical treatment. View Publication

BACKGROUND {\&} AIMS The nuclear receptor subfamily 0 group B member 2 (NR0B2,also called SHP) is expressed at high levels in the liver and intestine. Postprandial fibroblast growth factor 19 (human FGF19,mouse FGF15) signaling increases the transcriptional activity of SHP. We studied the functions of SHP and FGF19 in the intestines of mice,including their regulation of expression of the cholesterol transporter NPC1L1 )NPC1-like intracellular cholesterol transporter 1) and cholesterol absorption. METHODS We performed histologic and biochemical analyses of intestinal tissues from C57BL/6 and SHP-knockout mice and performed RNA-sequencing analyses to identify genes regulated by SHP. The effects of fasting and refeeding on intestinal expression of NPC1L1 were examined in C57BL/6,SHP-knockout,and FGF15-knockout mice. Mice were given FGF19 daily for 1 week; fractional cholesterol absorption,cholesterol and bile acid (BA) levels,and composition of BAs were measured. Intestinal organoids were generated from C57BL/6 and SHP-knockout mice,and cholesterol uptake was measured. Luciferase reporter assays were performed with HT29 cells. RESULTS We found that the genes that regulate lipid and ion transport in intestine,including NPC1L1,were up-regulated and that cholesterol absorption was increased in SHP-knockout mice compared with C57BL/6 mice. Expression of NPC1L1 was reduced in C57BL/6 mice after refeeding after fasting but not in SHP-knockout or FGF15-knockout mice. SHP-knockout mice had altered BA composition compared with C57BL/6 mice. FGF19 injection reduced expression of NPC1L1,decreased cholesterol absorption,and increased levels of hydrophilic BAs,including tauro-$\alpha$- and -$\beta$-muricholic acids; these changes were not observed in SHP-knockout mice. SREBF2 (sterol regulatory element binding transcription factor 2),which regulates cholesterol,activated transcription of NPC1L1. FGF19 signaling led to phosphorylation of SHP,which inhibited SREBF2 activity. CONCLUSIONS Postprandial FGF19 and SHP inhibit SREBF2,which leads to repression of intestinal NPC1L1 expression and cholesterol absorption. Strategies to increase FGF19 signaling to activate SHP might be developed for treatment of hypercholesterolemia. View Publication

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multitarget profile has,however,necessitated the application of combination therapies,which can pose major clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as new targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase-bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348,which are clinical PLK1 and JAK2-FLT3 kinase inhibitors,respectively,is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a new strategy for rational single-agent polypharmacological targeting. Furthermore,structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase-bromodomain inhibitors. View Publication

Timothy syndrome (TS) is a severe,multisystem disorder characterized by autism,epilepsy,long-QT syndrome and other neuropsychiatric conditions 1 . TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A,as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1,including delayed channel inactivation,prolonged depolarization-induced calcium rise,impaired interneuron migration,activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A 2 – 6 . We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and,following transplantation,in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed 7,we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons,suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly,these experiments illustrate how a multilevel,in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology. Subject terms: Autism spectrum disorders,Development of the nervous system View Publication

A broad range of hematopoietic stem cells and progenitors reside within a fraction of umbilical cord blood (UCB) that exhibits low light scatter properties (SSC(lo)) and high expression of aldehyde dehydrogenase (ALDH(br)). Many SSC(lo) ALDH(br) cells coexpress CD34; however,other cells express either ALDH or CD34. To investigate the developmental potential of these cell subsets,purified ALDH(br) CD34+,ALDH(neg) CD34+,and ALDH(br) CD34(neg) UCB cells were characterized within a variety of in vivo and in vitro assays. Primitive progenitors capable of multilineage development were monitored in long- and short-term repopulation assays performed on nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice,and in primary and secondary long-term culture assays. These progenitors were highly enriched within the ALDH(br) CD34+ fraction. This cell fraction also enriched short-term myeloid progenitors that were detected in vitro. By comparison,ALDH(neg) CD34+ cells contained few primitive progenitors and had diminished short-term myeloid potential but exhibited enhanced short-term natural killer (NK) cell development in vitro. The ALDH(br) CD34(neg) cells were not efficiently supported by any of the assays used. These studies suggested that in particular the expression of ALDH delineated distinct CD34+ stem cell and progenitor compartments. The differential expression of ALDH may provide a means to explore normal and malignant processes associated with myeloid and lymphoid development. View Publication

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein. The combination of biomarkers is crucial for AD diagnosis. The triggering receptor expressed on myeloid cells 2 (TREM2),a receptor expressed on microglia,is important in AD pathogenesis. Impairment of TREM2 function aggravates the toxic effects of amyloid plaques,and its activation has been shown to reduce Aβ burden and memory deficits. Increased levels of soluble TREM2 (sTREM2) in blood and cerebrospinal fluid is associated with AD. Therefore,TREM2 could serve as a non-invasive biomarker for AD. In this study,we developed a preclinical immunoassay to detect TREM2 for AD diagnosis. Highly sensitive and specific TREM2 antibodies were produced using the hybridoma technique. The three optimized immunoassays exhibited lower limit of quantitation (LLOQ) of 0.474,0.807,and 0.415 ng/mL,respectively. These preclinical immunoassays showed high sensitivity and specificity. The sandwich enzyme-linked immunosorbent assay (ELISA) could potentially be used for AD diagnosis. View Publication

By mimicking embryonic development of the hematopoietic system,we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines,extra cellular matrix components,and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% ± 16%,from seven pluripotent lines) from the differentiation culture,including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38- hematopoietic progenitors. Moreover,the numbers of hematopoietic progenitor cells generated,as measured by colony forming unit assays,were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors,it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability. View Publication

BackgroundHematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression,which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury,while if and how the niche is reshaped and regulates HSC regeneration are poorly understood.MethodsA mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number,distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry,immunofluorescence,colony assay and bone marrow transplantation,in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro,and was consolidated using megakaryocyte-specific knockout mice and transgenic mice.ResultsMegakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile,transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury,whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically,HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion,and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs,but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently,the delicate coordination between proliferation,mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly,punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury,representing a superior therapeutic approach for myelosuppression.ConclusionsOur study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-024-01651-5. View Publication

Regulatory T (Treg)-based cell therapy holds promise for autoimmune and inflammatory diseases,yet challenges remain regarding the functional stability and persistence of transferred Tregs. Here we engineer Tregs to express a partial agonist form of IL-2 (IL-2pa) to enhance persistence while avoiding toxicity from excessive signaling. Mouse Tregs expressing wild-type IL-2 (Tregs-IL2wt) have only a transient growth advantage,limited by toxicity from likely excessive signaling. By contrast,mouse Tregs-IL2pa exhibit sustained expansion,long-term survival in immunocompetent mice for over a year,and bystander expansion of endogenous Tregs. Tregs-IL2pa maintain a stable activated phenotype,Treg-specific demethylation,and a diverse TCR repertoire. In vivo,prophylactic transfer of Tregs-IL2pa ameliorates multi-organ autoimmunity in a Treg depletion-induced mouse autoimmune model. Lastly,compared with control Treg,human Tregs-IL2pa show enhanced survival in the IL-2-depleted environment of immune-deficient mice and improved control of xenogeneic graft-versus-host disease. Our results thus show that IL-2pa self-sufficiency enhances the stability,durability and efficacy of Treg therapies in preclinical settings. Subject terms: Cell delivery,Regulatory T cells,Autoimmune diseases,Interleukins View Publication