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ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial-mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness,we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers,we showed this to be the case in A549 cells,which harbor a Ras mutation,and in two other non-small-cell lung cancer cell lines,H1975 and H1650,driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover,treatment with hydroxycitrate phenocopied the effects of ACL KD,suggesting that the enzymatic activity of ACL was critical. Indeed,acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway,not the Ras-mitogen-activated protein kinase (MAPK) pathway,and that EMT can be reversed by PI3K inhibitors,we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling,and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells,ACL overexpression increased snail expression and stemness,both of which were reduced by ACL KD. Furthermore,ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally,ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function,namely its impact on cancer stemness in a broad range of genetically diverse cell types. View Publication

The advent of human induced pluripotent stem cell (hiPSC) technology has produced patient-specific hiPSC derived cardiomyocytes (hiPSC-CMs) that can be used as a platform to study cardiac diseases and to explore new therapies.The ability to genetically manipulate hiPSC-CMs not only is essential for identifying the structural and/or functional role of a protein but can also provide valuable information regarding therapeutic applications. In this chapter,we describe protocols for culture,maintenance,and cardiac differentiation of hiPSCs. Then,we provide a basic procedure to transduce hiPSC-CMs. View Publication

Lysophosphatidic acid (LPA; 1-acyl-sn-glycero-3-phosphate) is a platelet-derived lipid mediator that activates its own G-protein-coupled receptor to trigger phospholipase C-mediated Ca2+ mobilization and other effector pathways in numerous cell types. In this study we have examined the structural features of LPA that are important for activation of the Ca(2+)-mobilizing receptor in human A431 carcinoma cells,which show an EC50 for oleoyl-LPA as low as 0.2 nM. When the acyl chain at the sn-1 position is altered,the rank order of potency is oleoyl-LPA textgreater arachidonoyl-LPA textgreater linolenoyl-LPA textgreater linoleoyl-LPA textgreater stearoyl-LPA = palmitoyl-LPA textgreater myristoyl-LPA. The shorter-chain species,lauroyl- and decanoyl-LPA,show little or no activity. Ether-linked LPA (1-O-hexadecyl-sn-glycero-3-phosphate) is somewhat less potent than the corresponding ester-linked LPA; its stereoisomer is about equally active. Deletion of the glycerol backbone causes a 1000-fold decrease in potency. Replacement of the phosphate group in palmitoyl-LPA by a hydrogen- or methyl-phosphonate moiety results in complete loss of activity. A phosphonate analogue with a methylene group replacing the oxygen at sn-3 has strongly decreased activity. All three phosphonate analogues induce cell lysis at doses textgreater 15 microM. Similarly,the methyl and ethyl esters of palmitoyl-LPA are virtually inactive and become cytotoxic at micromolar doses. None of the LPA analogues tested has antagonist activity. Sphingosine 1-phosphate,a putative messenger with some structural similarities to LPA,elicits a transient rise in intracellular [Ca2+] only at micromolar doses; however,cross-desensitization experiments indicate that sphingosine 1-phosphate does not act through the LPA receptor. The results indicate that,although many features of the LPA structure are important for optimal activity,the phosphate group is most critical,suggesting that this moiety is directly involved in receptor activation. View Publication

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone,doxorubicin,and daunorubicin,reduces daunorubicin accumulation and retention,and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells. View Publication

The association between type 2 diabetes (T2D) pathogenesis and immune-mediated tissue damage and insulin resistance suggests that T2D patients might benefit from the suppression of pathogenic inflammation. Foxp3+ Treg cells are crucial suppressors of inflammation,but the differentiation of Foxp3+ Treg cells is not static and is subject to conversion into IL-17-producing Th17-like cells upon receiving external signals. In this study,we examined the production of IL-17 by Treg cells. Compared to non-T2D controls,T2D patients presented significantly higher levels of IL-17-expressing cells in both Foxp3- CD4 T cells and Foxp3+ Treg cells. The frequencies of IL-17-nonexpressing Foxp3+ Treg cells,on the other hand,were not changed. Interestingly,IL-17-expressing Foxp3+ Treg cells were mutually exclusive from IL-10-expressing and TGF-$\beta$-expressing Foxp3+ Treg cells,suggesting that multiple subpopulations exist within the Foxp3+ Treg cells from T2D patients. In T2D patients,the frequencies of IL-17-expressing Foxp3+ Treg cells were positively correlated with the body mass index (BMI) and the HbA1c levels of T2D patients. The frequencies of IL-10-expressing Treg cells,on the other hand,were inversely associated with the BMI of both non-T2D controls and T2D patients. In addition,the suppressive activity of Treg cells was significantly lower in T2D patients than in non-T2D controls. Together,our study uncovered a dysregulation in Foxp3+ Treg cells from T2D patients,characterized by high IL-17 expression and low suppression activity. View Publication

The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However,such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here,we present SNIPE,a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells,which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations. Genome editing often requires marker genes for selection of edited cells. Here,the authors present SNIPE,a marker-free method that selects cells based on DNA sequence,enabling precise enrichment of edited cells and applications from evolutionary research to the elimination of cancer cells. View Publication

Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels,differentiation intermediates were investigated. ES cells generated progeny expressing CD34,which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7,the CD34+ cells developed two types of B220+ CD34- lymphocytes: CD3- cytotoxic lymphocytes and CD19+ pre-B cells,and such lymphoid potential was highly enriched in the CD34+ population. Interestingly,the cytotoxic cells expressed the natural killer (NK) cell markers,such as NKR-P1,perforin,and granzymes,classified into two types,one of which showed target specificity of NK cells. Thus,ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells,and both myeloid and lymphoid cells seem to be derived from the CD34+ intermediate,on which VEGF may play an important role. View Publication

INTRODUCTION Bone marrow mesenchymal stem cells (BMSCs) have been extensively investigated from a perspective on cardiac regeneration therapy. The current study aimed to investigate the protective effect conferred by BMSCs in subacute myocardial injury,and to identify an appropriate BMSC reinfusion time. METHODS BMSCs were isolated from human bone marrow blood. Daunorubicin (DNR)-induced subacute myocardial models were subsequently established. The rats with DNR-induced subacute myocardial injury were injected with dexrazoxane (DZR) and/or BMSCs at varying time points,after which cardiac function was evaluated by assessing left ventricular ejection fraction (LVEF) and fraction shortening (FS). The myocardial structural changes were analyzed,after which the levels of CD3 and human leukocyte antigen DR (HLA-DR) were examined to further validate the mechanism by which BMSCs could influence subacute myocardial injury. RESULTS BMSCs combined with DZR treatment enhanced the cardiac function of rats with DNR-induced myocardial injury,as reflected by increased LVEF and FS. DNR-induced myocardial injuries were mitigated via the application of BMSCs combined with treatment of DZR,accompanied by diminished infiltration or vacuolization. Moreover,BMSCs were observed to alleviate infiltration of T lymphocyte and antigen-presenting cells,as evidenced by reduced expression of CD3 and HLA-DR. CONCLUSION Taken together,this study demonstrates that BMSCs could protect against DNR-induced myocardial injury,especially in the first three days of DNR administration. BMSCs combined with DZR exert a better therapeutic effect,but there are individual differences. View Publication

Autologous feeder cells have been developed by various methods to minimize the presence of xenogenic entities in human embryonic stem cell (hESC) cultures. However,there was no systematic comparison of supportive effects of the feeder cells on hESC growth,nor comparison to the supportive effects of various feeder-free culture systems and standard mouse feeder cells. In this study,we aimed to compare the supportive abilities of autologous feeders derived either directly from H9 hESCs (H9 dF) or from outgrowth of embryoid body predifferentiated in suspension from H9 hESCs (H9 ebF). Mouse feeder system and matrigel-mTeSR1 feeder-free system were used as controls. H9 ebF was found to secrete more basic fibroblast growth factor in the conditioned medium than H9 dF did. The undifferentiated state of H9 hESCs was sustained more stably on H9 ebF than on H9 dF,and the differentiation potential of H9 hESCs on H9 ebF was higher than on H9 dF. We concluded that H9 ebF was an optimal autologous feeder to maintain the long-term undifferentiated state of hESCs in our current culture system. This study helps to standardize the autologous culture of hESCs. It also suggests a more definite direction for future development of xeno-free culture system for hESCs. View Publication

AbstractIntroductionNovel strategies are needed to address the rising burden of osteoporosis and fragility fractures. High-intensity resistance and impact (HiRIT) exercise has shown benefit in improving bone density in postmenopausal women with osteoporosis/osteopenia. Whether HiRIT can enhance the therapeutic effects of osteoporosis pharmacotherapy has not been established. ROLEX-DUO is a randomised controlled trial designed to assess the efficacy of romosozumab on various bone and muscle outcomes in combination with different exercise interventions in women with postmenopausal osteoporosis/osteopenia.Methods and analysisROLEX-DUO is an 8-month randomised placebo-controlled trial conducted at two tertiary referral centres for patients with osteoporosis/osteopenia in Sydney,New South Wales,Australia. The study is implementing the combination of romosozumab or placebo with different forms of exercise in postmenopausal women with osteoporosis/osteopenia without recent fragility fracture (n=102). Eligible women will be randomised 1:1:1 into one of three groups: (1) romosozumab with supervised HiRIT,(2) romosozumab with unsupervised low-intensity exercise or (3) placebo with unsupervised low-intensity exercise. Co-primary outcomes are the mean percentage change in lumbar spine bone mineral density (BMD),and mean change in five times sit-to-stand test performance (seconds) at 8 months. Secondary/exploratory outcomes include BMD changes at the femoral neck,total hip and distal radius,three-dimensional dual-energy X-ray absorptiometry (DXA) hip outcomes,DXA-derived lean and fat mass,serum markers of bone turnover (procollagen type 1 peptide,C-telopeptide of type 1 collagen) and bone biomarkers (dickkopf-1),serum extracellular vesicle analyses,36-Item Short Form Survey (SF-36) quality-of-life scores,Menopause-Specific Quality Of Life (MENQOL) Questionnaire menopause symptom burden scores,number of falls and fractures. Mixed-effects models will be performed to compare longitudinal outcome results between groups using intention-to-treat analysis.Ethics and disseminationThe trial was approved by the Northern Sydney Local Health District Human Research Ethics Committee (2022/ETH01794,protocol V.8,dated 03 July 2024). Participants will provide written informed consent prior to inclusion. Findings will be disseminated via peer-reviewed journals,scientific conferences and summary reports to funding bodies.Trial registration numberACTRN12623000867695. View Publication

Helminthic infections modulate host immunity and may protect their hosts from developing immunological diseases like inflammatory bowel disease. Induction of regulatory T cells (Tregs) may be an important part of this protective process. Heligmosomoides polygyrus bakeri infection also promotes the production of the regulatory cytokines TGF-beta and IL-10 in the gut. In the intestines,TGF-beta helps induce regulatory T cells. This study used Foxp3/IL-10 double reporter mice to investigate the effect of TGF-beta on the differentiation of colon and mesenteric lymph node-derived murine Foxp3- IL-10- CD4+ T cells into their regulatory phenotypes. Foxp3- IL-10- CD4+ T cells from H. polygyrus bakeri-infected mice,as opposed to T cells from uninfected animals,cultured in vitro with TGF-beta and anti-CD3/CD28 mAb differentiated into Foxp3+ and/or IL-10+ T cells. The IL-10-producing T cells nearly all displayed CD25. Smad7 is a natural inhibitor of TGF-beta signaling. In contrast to gut T cells from uninfected mice,Foxp3- IL10- CD4+ T cells from H. polygyrus bakeri-infected mice displayed reduced Smad7 expression and responded to TGF-beta with Smad2/3 phosphorylation. The TGF-beta-induced Tregs that express IL-10 blocked colitis when transferred into the Rag/CD25- CD4+ T cell transfer model of inflammatory bowel disease. TGF-beta had a greatly diminished capacity to induce Tregs in H. polygyrus bakeri-infected transgenic mice with constitutively high T cell-specific Smad7 expression. Thus,infection with H. polygyrus bakeri causes down-modulation in Smad7 expression in intestinal CD4+ T cells,which allows the TGF-beta produced in response to the infection to induce the Tregs that prevent colitis. View Publication

Adipose tissue stem cells (ADSCs) would be an attractive autologous cell source. However,ADSCs require invasive procedures,and has potential complications. Recently,urine stem cells (USCs) have been proposed as an alternative stem cell source. In this study,we compared USCs and ADSCs collected from the same patients on stem cell characteristics and capacity to differentiate into various cell lineages to provide a useful guideline for selecting the appropriate type of cell source for use in clinical application. The urine samples were collected via urethral catheterization,and adipose tissue was obtained from subcutaneous fat tissue during elective laparoscopic kidney surgery from the same patient (n = 10). Both cells were plated for primary culture. Cell proliferation,colony formation,cell surface markers,immune modulation,chromosome stability and multi-lineage differentiation were analyzed for each USCs and ADSCs at cell passage 3,5,and 7. USCs showed high cell proliferation rate,enhanced colony forming ability,strong positive for stem cell markers expression,high efficiency for inhibition of immune cell activation compared to ADSCs at cell passage 3,5,and 7. In chromosome stability analysis,both cells showed normal karyotype through all passages. In analysis of multi-lineage capability,USCs showed higher myogenic,neurogenic,and endogenic differentiation rate,and lower osteogenic,adipogenic,and chondrogenic differentiation rate compared to ADSCs. Therefore,we expect that USC can be an alternative autologous stem cell source for muscle,neuron and endothelial tissue reconstruction instead of ADSCs. View Publication