Displaying publications 1 - 20 of 29 in total

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  1. Yong YK, Saeidi A, Tan HY, Rosmawati M, Enström PF, Batran RA, et al.
    Front Immunol, 2018;9:472.
    PMID: 29616020 DOI: 10.3389/fimmu.2018.00472
    Mucosal-associated invariant T (MAIT) cells, defined as CD161++TCR iVα7.2+ T cells, play an important role in the innate defense against bacterial infections, and their functionality is impaired in chronic viral infections. Here, we investigated the frequency and functional role of MAIT cells in chronic hepatitis B virus (HBV) infection. The peripheral CD3+CD161++TCR iVα7.2+ MAIT cells in chronic HBV-infected patients and healthy controls were phenotypically characterized based on CD57, PD-1, TIM-3, and CTLA-4, as well as HLA-DR and CD38 expression. The frequency of MAIT cells was significantly decreased among chronic HBV-infected individuals as compared to controls. Expression of CD57, PD-1, CTLA-4, as well as HLA-DR and CD38 on MAIT cells was significantly elevated in chronic HBV-infected individuals relative to controls. The percentage of T cell receptor (TCR) iVα7.2+ CD161+ MAIT cells did not correlate with HBV viral load but inversely with HLA-DR on CD4+ T cells and MAIT cells and with CD57 on CD8+ T cells suggesting that decrease of MAIT cells may not be attributed to direct infection by HBV but driven by HBV-induced chronic immune activation. The percentage and expression levels of PD-1 as well as CTLA-4 on MAIT cells inversely correlated with plasma HBV-DNA levels, which may suggest either a role for MAIT cells in the control of HBV infection or the effect of HBV replication in the liver on MAIT cell phenotype. We report that decrease of TCR iVα7.2+ MAIT cells in the peripheral blood and their functions were seemingly impaired in chronic HBV-infected patients likely because of the increased expression of PD-1.
  2. Yong YK, Tan HY, Saeidi A, Rosmawati M, Atiya N, Ansari AW, et al.
    Innate Immun, 2017 07;23(5):459-467.
    PMID: 28606013 DOI: 10.1177/1753425917714854
    Hepatitis B virus (HBV) infection is a major cause of chronic liver disease that may progress to liver cirrhosis and hepatocellular carcinoma. Host immune responses represent the key determinants of HBV clearance or persistence. Here, we investigated the role of the early activation marker, CD69 and effector cytokines, granzyme B (GrB) and IFN-γ in the exhaustion of innate-like TCR Vα7.2+CD4+T cells, in 15 individuals with chronic HBV (CHB) infection where six were HBV DNA+ and nine were HBV DNA-. The percentage of cytokine-producing T cells and MAIT cells were significantly perturbed in HBV patients relative to healthy controls (HCs). The intracellular expression of GrB and IFN-γ was significantly reduced in MAIT cells derived from HBV-infected patients as compared to HCs, and the levels correlated with the percentage and levels [mean fluorescence intensity (MFI)] of CD69 expression. The total expression of CD69 (iMFI) was lower in CHB patients as compared to HCs. The frequency of CD69+ cells correlated with the levels of cytokine expression (MFI), particularly in CHB patients as compared to HCs. In summary, the polyfunctionality of peripheral T cells was significantly reduced among CHB patients, especially in the TCR Vα7.2+CD4+T cells, and the levels of cytokine expression correlated with functional cytokine levels.
  3. Yong YK, Tan HY, Jen SH, Shankar EM, Natkunam SK, Sathar J, et al.
    J Transl Med, 2017 05 31;15(1):121.
    PMID: 28569153 DOI: 10.1186/s12967-017-1226-4
    BACKGROUND: Currently, several assays can diagnose acute dengue infection. However, none of these assays can predict the severity of the disease. Biomarkers that predicts the likelihood that a dengue patient will develop a severe form of the disease could permit more efficient patient triage and allows better supportive care for the individual in need, especially during dengue outbreaks.

    METHODS: We measured 20 plasma markers i.e. IFN-γ, IL-10, granzyme-B, CX3CL1, IP-10, RANTES, CXCL8, CXCL6, VCAM, ICAM, VEGF, HGF, sCD25, IL-18, LBP, sCD14, sCD163, MIF, MCP-1 and MIP-1β in 141 dengue patients in over 230 specimens and correlate the levels of these plasma markers with the development of dengue without warning signs (DWS-), dengue with warning signs (DWS+) and severe dengue (SD).

    RESULTS: Our results show that the elevation of plasma levels of IL-18 at both febrile and defervescence phase was significantly associated with DWS+ and SD; whilst increase of sCD14 and LBP at febrile phase were associated with severity of dengue disease. By using receiver operating characteristic (ROC) analysis, the IL-18, LBP and sCD14 were significantly predicted the development of more severe form of dengue disease (DWS+/SD) (AUC = 0.768, P 

  4. Yong YK, Tan HY, Saeidi A, Wong WF, Vignesh R, Velu V, et al.
    Front Microbiol, 2019;10:2789.
    PMID: 31921004 DOI: 10.3389/fmicb.2019.02789
    Tuberculosis (TB) treatment monitoring is paramount to clinical decision-making and the host biomarkers appears to play a significant role. The currently available diagnostic technology for TB detection is inadequate. Although GeneXpert detects total DNA present in the sample regardless live or dead bacilli present in clinical samples, all the commercial tests available thus far have low sensitivity. Humoral responses against Mycobacterium tuberculosis (Mtb) antigens are generally low, which precludes the use of serological tests for TB diagnosis, prognosis, and treatment monitoring. Mtb-specific CD4+ T cells correlate with Mtb antigen/bacilli burden and hence might serve as good biomarkers for monitoring treatment progress. Omics-based techniques are capable of providing a more holistic picture for disease mechanisms and are more accurate in predicting TB disease outcomes. The current review aims to discuss some of the recent advances on TB biomarkers, particularly host biomarkers that have the potential to diagnose and differentiate active TB and LTBI as well as their use in disease prognosis and treatment monitoring.
  5. Wadhwa M, Kang HN, Jivapaisarnpong T, WHO implementation workshop on guidelines on procedures and data requirements for changes to approved biotherapeutic products, Andalucia LR, Blades CDRZ, et al.
    Biologicals, 2020 May;65:50-59.
    PMID: 31959504 DOI: 10.1016/j.biologicals.2019.12.008
    The first global workshop on implementation of the WHO guidelines on procedures and data requirements for changes to approved biotherapeutic products adopted by the WHO Expert Committee in 2018 was held in June 2019. The workshop participants recognized that the principles based on sound science and the potential for risk, as described in the WHO Guidelines on post-approval changes, which constitute the global standard for product life-cycle management are providing clarity and helping national regulatory authorities in establishing guidance while improving time-lines for an efficient regulation of products. Consequently, the regulatory situation for post-approval changes and guideline implementation is changing but there is a disparity between different countries. While the guidelines are gradually being implemented in some countries and also being considered in other countries, the need for regional workshops and further training on post-approval changes was a common theme reiterated by many participants. Given the complexities relating to post-approval changes in different regions/countries, there was a clear understanding among all participants that an efficient approach for product life-cycle management at a national level is needed to ensure faster availability of high standard, safe and efficacious medicines to patients as per the World Health Assembly Resolution 67.21.
  6. Vimali J, Yong YK, Murugesan A, Tan HY, Zhang Y, Ashwin R, et al.
    Front Biosci (Landmark Ed), 2024 Mar 22;29(3):128.
    PMID: 38538288 DOI: 10.31083/j.fbl2903128
    BACKGROUND: Chronic viral infection results in impaired immune responses rendering viral persistence. Here, we compared the quality of T-cell responses among chronic hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV)-infected individuals by examining the levels of expression of selected immune activation and exhaustion molecules on circulating MAIT cells and Tfh cells.

    METHODS: Cytokines were measured using a commercial Bio-plex Pro Human Cytokine Grp I Panel 17-plex kit (BioRad, Hercules, CA, USA). Inflammation was assessed by measuring an array of plasma cytokines, and phenotypic alterations in CD4+ T cells including circulating Tfh cells, CD8+ T cells, and TCR iVα7.2+ MAIT cells in chronic HBV, HCV, and HIV-infected patients and healthy controls. The cells were characterized based on markers pertaining to immune activation (CD69, ICOS, and CD27) proliferation (Ki67), cytokine production (TNF-α, IFN-γ) and exhaustion (PD-1). The cytokine levels and T cell phenotypes together with cell markers were correlated with surrogate markers of disease progression.

    RESULTS: The activation marker CD69 was significantly increased in CD4+hi T cells, while CD8+ MAIT cells producing IFN-γ were significantly increased in chronic HBV, HCV and HIV infections. Six cell phenotypes, viz., TNF-α+CD4+lo T cells, CD69+CD8+ T cells, CD69+CD4+ MAIT cells, PD-1+CD4+hi T cells, PD-1+CD8+ T cells, and Ki67+CD4+ MAIT cells, were independently associated with decelerating the plasma viral load (PVL). TNF-α levels showed a positive correlation with increase in cytokine levels and decrease in PVL.

    CONCLUSION: Chronic viral infection negatively impacts the quality of peripheral MAIT cells and Tfh cells via differential expression of both activating and inhibitory receptors.

  7. Tham HY, Song AA, Yusoff K, Tan GH
    Biotechniques, 2020 09;69(3):161-170.
    PMID: 32787565 DOI: 10.2144/btn-2020-0034
    Endolysins have been studied intensively as an alternative to antibiotics. In this study, endolysin derived from a phage which infects methicillin-resistant Staphylococcus aureus (MRSA) was cloned and expressed in Escherichia coli pET28a. Initially, the endolysin was cloned using BamHI/XhoI, resulting in expression of a recombinant endolysin which was expressed in inclusion bodies. While solubilization was successful, the protein remained nonfunctional. Recloning the endolysin using NcoI/XhoI resulted in expression of soluble and functional proteins at 18°C. The endolysin was able to form halo zones on MRSA plates and showed a reduction in turbidity of MRSA growth. Therefore, cloning strategies should be chosen carefully even in an established expression system as they could greatly affect the functionality of the expressed protein.
  8. Selvavinayagam ST, Suvaithenamudhan S, Yong YK, Hemashree K, Rajeshkumar M, Kumaresan A, et al.
    J Med Virol, 2024 Feb;96(2):e29456.
    PMID: 38329187 DOI: 10.1002/jmv.29456
    A state-wide prospective longitudinal investigation of the genomic surveillance of the omicron B.1.1.529 SARS-CoV-2 variant and its sublineages in Tamil Nadu, India, was conducted between December 2021 and March 2023. The study aimed to elucidate their mutational patterns and their genetic interrelationship in the Indian population. The study identified several unique mutations at different time-points, which likely could attribute to the changing disease characteristics, transmission, and pathogenicity attributes of omicron variants. The study found that the omicron variant is highly competent in its mutating potentials, and that it continues to evolve in the general population, likely escaping from natural as well as vaccine-induced immune responses. Our findings suggest that continuous surveillance of viral variants at the global scenario is warranted to undertake intervention measures against potentially precarious SARS-CoV-2 variants and their evolution.
  9. Saeidi A, Chong YK, Yong YK, Tan HY, Barathan M, Rajarajeswaran J, et al.
    Cell Immunol, 2015 Sep;297(1):19-32.
    PMID: 26071876 DOI: 10.1016/j.cellimm.2015.05.005
    The role of T-cell immunosenescence and functional CD8(+) T-cell responses in HIV/TB co-infection is unclear. We examined and correlated surrogate markers of HIV disease progression with immune activation, immunosenescence and differentiation using T-cell pools of HIV/TB co-infected, HIV-infected and healthy controls. Our investigations showed increased plasma viremia and reduced CD4/CD8 T-cell ratio in HIV/TB co-infected subjects relative to HIV-infected, and also a closer association with changes in the expression of CD38, a cyclic ADP ribose hydrolase and CD57, which were consistently expressed on late-senescent CD8(+) T cells. Up-regulation of CD57 and CD38 were directly proportional to lack of co-stimulatory markers on CD8(+) T cells, besides diminished expression of CD127 (IL-7Rα) on CD57(+)CD4(+) T cells. Notably, intracellular IFN-γ, perforin and granzyme B levels in HIV-specific CD8(+) T cells of HIV/TB co-infected subjects were diminished. Intracellular CD57 levels in HIV gag p24-specific CD8(+) T cells were significantly increased in HIV/TB co-infection. We suggest that HIV-TB co-infection contributes to senescence associated with chronic immune activation, which could be due to functional insufficiency of CD8(+) T cells.
  10. Saeidi A, Tien Tien VL, Al-Batran R, Al-Darraji HA, Tan HY, Yong YK, et al.
    PLoS One, 2015;10(4):e0124659.
    PMID: 25894562 DOI: 10.1371/journal.pone.0124659
    Mucosal-associated invariant T (MAIT) cells are evolutionarily conserved antimicrobial MR1-restricted CD8(+) T cells co-expressing the semi-invariant TCR Vα7.2, and are numerous in the blood and mucosal tissues of humans. MAIT cells appear to undergo exhaustion in chronic viral infections. However, their role in human immunodeficiency virus type 1 (HIV-1) mono-infection and HIV/tuberculosis (TB) co-infection have seldom been elaborately investigated. We conducted a cross-sectional study to investigate the frequencies and phenotypes of CD161(++)CD8(+) T cells among anti-retroviral therapy (ART)/anti-TB therapy (ATT) treatment-naïve HIV/TB co-infected, ART/TB treated HIV/TB co-infected, ART naïve HIV-infected, ART-treated HIV-infected patients, and HIV negative healthy controls (HCs) by flow cytometry. Our data revealed that the frequency of MAIT cells was severely depleted in HIV mono- and HIV/TB co-infections. Further, PD-1 expression on MAIT cells was significantly increased in HIV mono- and HIV-TB co-infected patients. The frequency of MAIT cells did not show any significant increase despite the initiation of ART and/or ATT. Majority of the MAIT cells in HCs showed a significant increase in CCR6 expression as compared to HIV/TB co-infections. No marked difference was seen with expressions of chemokine co-receptor CCR5 and CD103 among the study groups. Decrease of CCR6 expression appears to explain why HIV-infected patients display weakened mucosal immune responses.
  11. Saeidi A, Ellegård R, Yong YK, Tan HY, Velu V, Ussher JE, et al.
    J Leukoc Biol, 2016 08;100(2):305-14.
    PMID: 27256572 DOI: 10.1189/jlb.4RU0216-084R
    MAIT cells represent an evolutionarily conserved, MR1-restricted, innate-like cell subset that express high levels of CD161; have a canonical semi-invariant TCR iVα7.2; and may have an important role in mucosal immunity against various bacterial and fungal pathogens. Mature MAIT cells are CD161(hi)PLZF(hi)IL-18Rα(+)iVα7.2(+)γδ-CD3(+)CD8(+) T cells and occur in the peripheral blood, liver, and mucosa of humans. MAIT cells are activated by a metabolic precursor of riboflavin synthesis presented by MR1 and, therefore, respond to many bacteria and some fungi. Despite their broad antibacterial properties, their functional role in persistent viral infections is poorly understood. Although there is an increasing line of evidence portraying the depletion of MAIT cells in HIV disease, the magnitude and the potential mechanisms underlying such depletion remain unclear. Recent studies suggest that MAIT cells are vulnerable to immune exhaustion as a consequence of HIV and hepatitis C virus infections and HIV/tuberculosis coinfections. HIV infection also appears to cause functional depletion of MAIT cells resulting from abnormal expression of T-bet and EOMES, and effective ART is unable to completely salvage functional MAIT cell loss. Depletion and exhaustion of peripheral MAIT cells may affect mucosal immunity and could increase susceptibility to opportunistic infections during HIV infection. Here, we review some of the important mechanisms associated with depletion and functional loss of MAIT cells and also suggest potential immunotherapeutic strategies to restore MAIT cell functions, including the use of IL-7 to restore effector functions in HIV disease.
  12. Preeyaa SU, Murugesan A, Sopnajothi S, Yong YK, Tan HY, Larsson M, et al.
    Viral Immunol, 2020 11;33(9):610-615.
    PMID: 32996843 DOI: 10.1089/vim.2020.0149
    Peripheral follicular helper T (pTfh) cells represent specialized CD4+ T cells that help B cells to secrete antibodies. Dengue infection appears to cause immune activation in a wide array of immune cells. Herein, we investigated the signatures of immune activation of circulating Tfh cells and mucosal-associated invariant T (MAIT) cells in adult subjects with confirmed acute clinical dengue virus (DENV) infection by multiparametric flow cytometry. The acute DENV infection induced a significant expansion of highly activated pTfh cells and circulating MAIT cells during acute febrile infection. We found a higher frequency of activated PD-1+ Tfh cells and CD38+ pTfh cells in clinical DENV infection. We also found similar activated and expanding phenotypes of MAIT cells in the patients tested. The total counts of activated pTfh cells and circulating MAIT cells were higher in dengue patients relative to healthy controls. We concluded that pTfh cells and circulating MAIT cells represent activated phenotypes in acute DENV infection.
  13. M Yatim H, Wong YY, Neoh CF, Lim SH, Hassali MA, Hong YH
    Public Health, 2019 Aug;173:5-8.
    PMID: 31207425 DOI: 10.1016/j.puhe.2019.04.020
    OBJECTIVE: The objective of this study was to explore factors influencing patients with hypertension to participating in a hypertension self-management education (HSME) programme and challenges of sustaining the learnt self-care practices.

    STUDY DESIGN: This was a qualitative study with focus group discussions.

    METHODS: Focus group discussions using a semistructured moderator guide were conducted among participants who had attended the HSME programme. Data were audio recorded, transcribed verbatim and analysed using a thematic analysis approach.

    RESULTS: Three focus groups involving 19 participants were conducted. Four major themes emerged from the data collected. Most participants enjoyed the group-based HSME sessions because sharing experiences with those having similar health problems can reduce their sense of isolation. However, the participants highlighted the difficulty in sustaining self-care practices in the presence of friends and family influences.

    CONCLUSION: A number of patient-, family- and community-level motivators and barriers to patients' hypertension self-management have been identified. Efforts to tailor behavioural interventions to sustain daily self-care activities during social and cultural events are imperative.
  14. Liu X, Yang X, Shao J, Hong Y, Gopinath SCB, Chen Y, et al.
    J Anal Methods Chem, 2020;2020:6528572.
    PMID: 32309010 DOI: 10.1155/2020/6528572
    Cervical, ovarian, and endometrial cancers are common in the female reproductive system. Cervical cancer starts from the cervix, while ovarian cancer develops when abnormal cells grow in the ovary. Endometrial or uterine cancer starts from the lining of the womb in the endometrium. Approximately 12,000 women are affected every year by cervical cancer in the United States. Squamous cell carcinoma antigen (SCC-Ag) is a well-established biomarker in serum for diagnosing gynecological cancers, and its levels were observed to be elevated in cervical, ovarian, and endometrial cancer patients. Moreover, SCC-Ag was used to identify the tumor size and progression stages. Various biosensing systems have been proposed to identify SCC-Ag; herein, enhanced interdigitated electrode sensing is presented with the use of gold nanoparticles (GNPs) to conjugate an antigen/antibody. It was proved that the limit of detection is 62.5 fM in the case of antibody-GNP, which is 2-fold higher than that by SCC-Ag-GNP. Furthermore, the antibody-GNP-modified surface displays greater current increases with concomitant dose-dependent SCC-Ag levels. High analytical performance was shown by the discrimination against α-fetoprotein and CYFRA 21-1 at 1 pM. An enhanced sensing system is established for gynecological tumors, representing an advance from the earlier detection methods.
  15. Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, et al.
    Autophagy, 2016;12(1):1-222.
    PMID: 26799652 DOI: 10.1080/15548627.2015.1100356
  16. Katsos N, Cummins C, Ezeizabarrena MJ, Gavarró A, Kuvač Kraljević J, Hrzica G, et al.
    Proc Natl Acad Sci U S A, 2016 08 16;113(33):9244-9.
    PMID: 27482119 DOI: 10.1073/pnas.1601341113
    Learners of most languages are faced with the task of acquiring words to talk about number and quantity. Much is known about the order of acquisition of number words as well as the cognitive and perceptual systems and cultural practices that shape it. Substantially less is known about the acquisition of quantifiers. Here, we consider the extent to which systems and practices that support number word acquisition can be applied to quantifier acquisition and conclude that the two domains are largely distinct in this respect. Consequently, we hypothesize that the acquisition of quantifiers is constrained by a set of factors related to each quantifier's specific meaning. We investigate competence with the expressions for "all," "none," "some," "some…not," and "most" in 31 languages, representing 11 language types, by testing 768 5-y-old children and 536 adults. We found a cross-linguistically similar order of acquisition of quantifiers, explicable in terms of four factors relating to their meaning and use. In addition, exploratory analyses reveal that language- and learner-specific factors, such as negative concord and gender, are significant predictors of variation.
  17. Huang Z, Aweya JJ, Zhu C, Tran NT, Hong Y, Li S, et al.
    Front Immunol, 2020;11:574721.
    PMID: 33224140 DOI: 10.3389/fimmu.2020.574721
    Aquaculture production of crustaceans (mainly shrimp and crabs) has expanded globally, but disease outbreaks and pathogenic infections have hampered production in the last two decades. As invertebrates, crustaceans lack an adaptive immune system and mainly defend and protect themselves using their innate immune system. The immune system derives energy and metabolites from nutrients, with amino acids constituting one such source. A growing number of studies have shown that amino acids and their metabolites are involved in the activation, synthesis, proliferation, and differentiation of immune cells, as well as in the activation of immune related signaling pathways, reduction of inflammatory response and regulation of oxidative stress. Key enzymes in amino acid metabolism have also been implicated in the regulation of the immune system. Here, we reviewed the role played by amino acids and their metabolites in immune-modulation in crustaceans. Information is inferred from mammals and fish where none exists for crustaceans. Research themes are identified and the relevant research gaps highlighted for further studies.
  18. Hong Y, Hassan N, Cheah YK, Jalaludin MY, Kasim ZM
    Malays Fam Physician, 2017;12(2):18-22.
    PMID: 29423125
    The Clinical Practice Guidelines on the Management of Type 1 Diabetes Mellitus in Children & Adolescents was developed by a multidisciplinary development group and approved by the Ministry of Health Malaysia in 2015. A systematic review of 15 clinical questions was conducted using the evidence retrieved mainly from MEDLINE and Cochrane databases. Critical appraisal was done using the Critical Appraisal Skills. Recommendations were formulated on the accepted 136 evidences using the principles of Grading Recommendations, Assessment, Development and Evaluation tailored to the local setting. Type 1 diabetes mellitus is a chronic disease, which usually occurs at an early age, and is associated with various complications including retinopathy, nephropathy, neuropathy and cardiovascular morbidity. Good glycaemic control early in the disease results in lower frequency of chronic diabetes complications, which in turn reduces the healthcare cost. Accurate classification of diabetes and optimum management with the aim to achieve glycaemic targets is of utmost importance.
  19. Hong Y, Al Mamun A, Yang Q, Masukujjaman M
    Sci Rep, 2024 Jan 19;14(1):1706.
    PMID: 38243057 DOI: 10.1038/s41598-024-52215-z
    The fashion industry has a significant impact on the environment, and sustainable fashion consumption (SFC) has become a pressing concern. This study aimed to investigate the factors influencing sustainable fashion consumption behavior (SCB) among Chinese adults, specifically the role of values, attitudes, and norms in shaping such behavior, using the value-belief-norm framework. The study used an online cross-sectional survey design to collect data from 350 participants recruited through a convenience sampling method using social media platforms and email invitations, and the obtained data were analyzed using partial least squares structural equation modelling. The results of the study showed that biospheric (BV), altruistic (AV), and egoistic (EV) values significantly influenced the New ecological paradigm (EP), which, in turn, positively affected awareness of consequences (AC). Personal norms (PN) were positively influenced by EP, AC, and ascription of responsibility (AR). Social norms (SN) and trust in recycling (TR) were also found to positively influence sustainable fashion consumption intentions (SCI). Finally, the study found that SCI and TR were significant predictors of SCB, whereas the moderating effect of TR not statistically significant. The study's originality lies in its comprehensive investigation of the interplay between various factors (particularly using norms in two facets; PN and SN) in shaping SCB, using a structural equation modeling approach, and exploring the moderating effect of TR. The findings of this study suggest that interventions aimed at promoting SFC should focus on fostering values and beliefs that prioritize the environment, encouraging individuals to take responsibility for their actions, creating an environment in which SFC is normalized, and increasing TR.
  20. Ho SY, Goh CW, Gan JY, Lee YS, Lam MK, Hong N, et al.
    Zebrafish, 2014 Oct;11(5):407-20.
    PMID: 24967707 DOI: 10.1089/zeb.2013.0879
    Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.
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