Displaying all 18 publications

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  1. Comments on "Therapy with mesenchymal stromal cells or conditioned medium reverse cardiac alterations in a high-fat diet-induced obesity model"
    Gnanasegaran N
    Cytotherapy, 2017 12;19(12):1551.
    PMID: 29033284 DOI: 10.1016/j.jcyt.2017.09.002
  2. Differentiation of stem cells derived from carious teeth into dopaminergic-like cells
    Gnanasegaran N, Govindasamy V, Abu Kasim NH
    Int Endod J, 2016 Oct;49(10):937-49.
    PMID: 26354006 DOI: 10.1111/iej.12545
    AIM: To investigate whether dental pulp stem cells from carious teeth (DPSCs-CT) can differentiate into functional dopaminergic-like (DAergic) cells and provide an alternative cell source in regenerative medicine.

    METHODOLOGY: Dental pulp stem cells from healthy (DPSCs) and carious teeth (DPSCs-CT) were isolated from young donors. Both cell lines were expanded in identical culture conditions and subsequently differentiated towards DAergic-like cells using pre-defined dopaminergic cocktails. The dopaminergic efficiencies were evaluated both at gene and protein as well as at secretome levels.

    RESULTS: The efficiency of DPSCs-CT to differentiate into DAergic-like cells was not equivalent to that of DPSCs. This was further reflected in both gene and protein generation whereby key neuronal markers such as nestin, NURR1 and beta-III-tubulin were expressed significantly lower as compared to differentiated DPSCs (P 

  3. Fulltext ReNCell VM conditioned medium enhances the induction of dental pulp stem cells into dopaminergic like cells
    Gnanasegaran N, Govindasamy V, Musa S, Abu Kasim NH
    Cytotechnology, 2016 Mar;68(2):343-53.
    PMID: 25322895 DOI: 10.1007/s10616-014-9787-z
    Among the debilitating diseases, neurological related diseases are the most challenging ones to be treated using cell replacement therapies. Recently, dental pulp stem cells (SHED) were found to be most suitable cell choice for neurological related diseases as evidenced with many preclinical studies. To enhance the neurological potential of SHED, we recapitulated one of the pharmacological therapeutic tools in cell replacement treatment, we pre-conditioned dental pulp stem cells (SHED) with culture medium of ReNCell VM, an immortalized neuron progenitor cell, prior to neurogenesis induction and investigated whether this practice enhances their neurogenesis potential especially towards dopaminergic neurons. We hypothesed that the integration of pharmacological practices such as co-administration of various drugs, a wide range of doses and duration as well as pre-conditioning into cell replacement may enhance the efficacy of stem cell therapy. In particular, pre-conditioning is shown to be involved in the protective effect from some membrano-tropic drugs, thereby improving the resistance of cell structures and homing capabilities. We found that cells pre-treated with ReNCell VM conditioned medium displayed bipolar structures with extensive branches resembling putative dopaminergic neurons as compared to non-treated cells. Furthermore, many neuronal related markers such as NES, NR4A2, MSI1, and TH were highly expressed (fold changes > 2; p 
  4. Fulltext Different isolation methods alter the gene expression profiling of adipose derived stem cells
    Gnanasegaran N, Govindasamy V, Musa S, Kasim NH
    Int J Med Sci, 2014;11(4):391-403.
    PMID: 24669199 DOI: 10.7150/ijms.7697
    Human adipose stem cells (ASCs) has been in the limelight since its discovery as a suitable source of mesenchymal stem cells (MSCs) in regenerative medicine. Currently, two major techniques are used to isolate ASCs, namely liposuction and tissue biopsy. These two methods are relatively risk-free but the question as to which method could give a more efficient output remains unclear. Thus, this study was carried out to compare and contrast the output generated in regards to growth kinetics, differentiation capabilities in vitro, and gene expression profiling. It was found that ASCs from both isolation methods were comparable in terms of growth kinetics and tri-lineage differentiation. Furthermore, ASCs from both populations were reported as CD44(+), CD73(+), CD90(+), CD166(+), CD34(-), CD45(-) and HLA-DR(-). However, in regards to gene expression, a group of overlapping genes as well as distinct genes were observed. Distinct gene expressions indicated that ASCs (liposuction) has endoderm lineage propensity whereas ASCs (biopsy) has a tendency towards mesoderm/ectoderm lineage. This information suggests involvement in different functional activity in accordance to isolation method. In conclusion, future studies to better understand these gene functions should be carried out in order to contribute in the applicability of each respective cells in regenerative therapy.
  5. Comparison of immunodulatory properties of dental pulp stem cells derived from healthy and inflamed teeth
    Yazid FB, Gnanasegaran N, Kunasekaran W, Govindasamy V, Musa S
    Clin Oral Investig, 2014 Dec;18(9):2103-12.
    PMID: 24549764 DOI: 10.1007/s00784-014-1207-4
    The aim of this study was to investigate the immunodulatory properties of dental pulp stem cells derived from healthy (SCD) and inflamed pulp deciduous (SCDIP) tissues. The overall hypothesis is that SCDIP possess equal immune properties with SCD and could be used as an alternative tissue source in regenerative medicine.
  6. Method for Evaluating Neuromodulatory Properties of Dental Pulp Stem Cell as an In Vitro Model for Parkinson's Disease
    Gnanasegaran N, Thimiri Govinda Raj DB, Arumugam S
    Methods Mol Biol, 2020;2125:193-196.
    PMID: 31489601 DOI: 10.1007/7651_2019_261
    Several research groups have utilized dental pulp stem cells for numerous studies as treatment modality for Parkinson's disease (PD). However, the roles of dental pulp stem cells in governing the Parkinson's disease inflammatory microenvironment remain to be evaluated. In this article, we elaborate the method where we can investigate the effects of dental pulp stem cells on neurons and microglia in an in vitro inflammatory microenvironment.
  7. Neuroimmunomodulatory properties of DPSCs in an in vitro model of Parkinson's disease
    Gnanasegaran N, Govindasamy V, Mani V, Abu Kasim NH
    IUBMB Life, 2017 09;69(9):689-699.
    PMID: 28685937 DOI: 10.1002/iub.1655
    In neurodegenerative diseases, such as Alzheimer's and Parkinson's, microglial cell activation is thought to contribute to their degeneration by producing neurotoxic compounds. While dental pulp stem cells (DPSCs) have been regarded as the next possible cell source for cell replacement therapy (CRT), their actual role when exposed in such harsh environment remains elusive. In this study, the immunomodulatory behavior of DPSCs from human subjects was investigated in a coculture system consisting of neuron and microglia which were treated with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine, which mimics the inflammatory conditions and contribute to degeneration of dopaminergic (DA-ergic) neurons. Assessments were performed on their proliferation, extent of DNA damage, productions of reactive oxygen species (ROS) and nitric oxide (NO), as well as secretion of inflammatory mediators. Notably, DPSCs were shown to attenuate their proliferation, production of ROS, and NO significantly (P 
  8. Effects of cell cycle phases on the induction of dental pulp stem cells toward dopaminergic-like cells
    Gnanasegaran N, Govindasamy V, Kathirvaloo P, Musa S, Abu Kasim NH
    J Tissue Eng Regen Med, 2018 02;12(2):e881-e893.
    PMID: 28079995 DOI: 10.1002/term.2401
    Parkinson's disease (PD) is characterized by tremors and cognitive issues, and is due to the death of dopaminergic (DA-ergic) neurons in brain circuits that are responsible for producing neurotransmitter dopamine (DA). Currently, cell replacement therapies are underway to improve upon existing therapeutic approaches such as drug treatments and electrical stimulation. Among the widely available sources, dental pulp stem cells (DPSCs) from deciduous teeth have gained popularity because of their neural crest origin and inherent propensity toward neuronal lineage. Despite the various pre-clinical studies conducted, an important factor yet to be elucidated is the influence of growth phases in a typical trans-differentiation process. This study selected DPSCs at three distinct time points with variable growth phase proportions (G0/G1, S and G2/M) for in vitro trans-differentiation into DA-ergic-like cells. Using commercially available PCR arrays, we identified distinct gene profiles pertaining to cell cycles in these phases. The differentiation outcomes were assessed in terms of morphology and gene and protein expression, as well as with functional assays. It was noted that DPSCs with the highest G0/G1 phase were comparatively the best, representing at least a 2-fold up regulation (p 
  9. Fulltext Differential expression of basal microRNAs' patterns in human dental pulp stem cells
    Vasanthan P, Govindasamy V, Gnanasegaran N, Kunasekaran W, Musa S, Abu Kasim NH
    J Cell Mol Med, 2015 Mar;19(3):566-80.
    PMID: 25475098 DOI: 10.1111/jcmm.12381
    MicroRNAs (miRNAs) are small non-coding RNAs that regulate translation of mRNA into protein and play a crucial role for almost all biological activities. However, the identification of miRNAs from mesenchymal stem cells (MSCs), especially from dental pulp, is poorly understood. In this study, dental pulp stem cells (DPSCs) were characterized in terms of their proliferation and differentiation capacity. Furthermore, 104 known mature miRNAs were profiled by using real-time PCR. Notably, we observed 19 up-regulated miRNAs and 29 significantly down-regulated miRNAs in DPSCs in comparison with bone marrow MSCs (BM-MSCs). The 19 up-regulated miRNAs were subjected to ingenuity analysis, which were composed into 25 functional networks. We have chosen top 2 functional networks, which comprised 10 miRNA (hsa-miR-516a-3p, hsa-miR-125b-1-3p, hsa-miR-221-5p, hsa-miR-7, hsa-miR-584-5p, hsa-miR-190a, hsa-miR-106a-5p, hsa-mir-376a-5p, hsa-mir-377-5p and hsa-let-7f-2-3p). Prediction of target mRNAs and associated biological pathways regulated by each of this miRNA was carried out. We paid special attention to hsa-miR-516a-3p and hsa-miR-7-5p as these miRNAs were highly expressed upon validation with qRT-PCR analysis. We further proceeded with loss-of-function analysis with these miRNAs and we observed that hsa-miR-516a-3p knockdown induced a significant increase in the expression of WNT5A. Likewise, the knockdown of hsa-miR-7-5p increased the expression of EGFR. Nevertheless, further validation revealed the role of WNT5A as an indirect target of hsa-miR-516a-3p. These results provide new insights into the dynamic role of miRNA expression in DPSCs. In conclusion, using miRNA signatures in human as a prediction tool will enable us to elucidate the biological processes occurring in DPSCs.
  10. Fulltext Diverse effects of lead nitrate on the proliferation, differentiation, and gene expression of stem cells isolated from a dental origin
    Abdullah M, Rahman FA, Gnanasegaran N, Govindasamy V, Abu Kasim NH, Musa S
    ScientificWorldJournal, 2014;2014:235941.
    PMID: 24616615 DOI: 10.1155/2014/235941
    Lead (Pb(2+)) exposure continues to be a significant public health problem. Therefore, it is vital to have a continuous epidemiological dataset for a better understanding of Pb(2+) toxicity. In the present study, we have exposed stem cells isolated from deciduous and permanent teeth, periodontal ligament, and bone marrow to five different types of Pb(2+) concentrations (160, 80, 40, 20, and 10 µM) for 24 hours to identify the adverse effects of Pb(2+) on the proliferation, differentiation, and gene expression on these cell lines. We found that Pb(2+) treatment altered the morphology and adhesion of the cells in a dose-dependent manner. There were no significant changes in terms of cell surface phenotypes. Cells exposed to Pb(2+) continued to differentiate into chondrogenesis and adipogenesis, and a severe downregulation was observed in osteogenesis. Gene expression studies revealed a constant expression of key markers associated with stemness (Oct 4, Rex 1) and DNA repair enzyme markers, but downregulation occurred with some ectoderm and endoderm markers, demonstrating an irregular and untimely differentiation trail. Our study revealed for the first time that Pb(2+) exposure not only affects the phenotypic characteristics but also induces significant alteration in the differentiation and gene expression in the cells.
  11. Unique molecular signatures influencing the biological function and fate of post-natal stem cells isolated from different sources
    Abu Kasim NH, Govindasamy V, Gnanasegaran N, Musa S, Pradeep PJ, Srijaya TC, et al.
    J Tissue Eng Regen Med, 2015 Dec;9(12):E252-66.
    PMID: 23229816 DOI: 10.1002/term.1663
    The discovery of mesenchymal stem cells (MSCs) from a myriad of tissues has triggered the initiative of establishing tailor-made stem cells for disease-specific therapy. Nevertheless, lack of understanding on the inherent differential propensities of these cells may restrict their clinical outcome. Therefore, a comprehensive study was done to compare the proliferation, differentiation, expression of cell surface markers and gene profiling of stem cells isolated from different sources, viz. bone marrow, Wharton's jelly, adipose tissue and dental pulp. We found that although all MSCs were phenotypically similar to each other, Wharton's jelly (WJ) MSCs and dental pulp stem cells (DPSCs) were highly proliferative as compared to bone marrow (BM) MSCs and adipose tissue (AD) MSCs. Moreover, indistinguishable cell surface characteristics and differentiation capacity were confirmed to be similar among all cell types. Based on gene expression profiling, we postulate that BM-MSCs constitutively expressed genes related to inflammation and immunodulation, whereas genes implicated in tissue development were highly expressed in AD-MSCs. Furthermore, the transcriptome profiling of WJ-MSCs and DPSCs revealed an inherent bias towards the neuro-ectoderm lineage. Based on our findings, we believe that there is no unique master mesenchymal stem cell that is appropriate to treat all target diseases. More precisely, MSCs from different sources exhibit distinct and unique gene expression signatures that make them competent to give rise to specific lineages rather than others. Therefore, stem cells should be subjected to rigorous characterization and utmost vigilance needs to be adopted in order to choose the best cellular source for a particular disease.
  12. Expression patterns of immune genes in long-term cultured dental stem cells
    Jayaraman P, Govindasamy V, Gnanasegaran N, Kunasekaran W, Vasanthan P, Musa S, et al.
    Clin Oral Investig, 2016 Jan;20(1):109-16.
    PMID: 26048030 DOI: 10.1007/s00784-015-1497-1
    Long-term culture system is used to prevent the impediment of insufficient cells and is good for low starting materials such as dental pulp or periodontal ligament. In general, although cell viability and functionality are the most common aspects taken into consideration in culturing cells for a long term, they may not truly represent the biological state of the cells. Hence, we explored the behaviour of another important aspect which is the immune properties in long-term cultured cells.
  13. Angiogenic effect of platelet-rich concentrates on dental pulp stem cells in inflamed microenvironment
    Bindal P, Gnanasegaran N, Bindal U, Haque N, Ramasamy TS, Chai WL, et al.
    Clin Oral Investig, 2019 Oct;23(10):3821-3831.
    PMID: 30687907 DOI: 10.1007/s00784-019-02811-5
    OBJECTIVE: In this study, we aimed to determine the suitable concentrations of human platelet lysate (HPL) and platelet-rich plasma (PRP) for maintaining the in vitro proliferative and angiogenic potential of inflamed dental pulp stem cells.

    MATERIALS AND METHODS: Lipopolysaccharide (LPS)-induced inflamed dental pulp-derived stem cells (iDPSCs) were treated with different concentrations of HPL and PRP (10% and 20%) followed by determination of viability using Alamar Blue assay. Expression of angiogenesis-, adhesion-, and inflammation-regulating genes was also analyzed using RT-qPCR array. Furthermore, expression of growth factors at protein level in the cell culture microenvironment was measured using multiplex assay.

    RESULTS: Viability of iDPSCs was significantly (p 

  14. Immune responses of human dental pulp stem cells in lipopolysaccharide-induced microenvironment
    Bindal P, Ramasamy TS, Kasim NHA, Gnanasegaran N, Chai WL
    Cell Biol Int, 2018 Jul;42(7):832-840.
    PMID: 29363846 DOI: 10.1002/cbin.10938
    This study aimed to investigate the effect of inflammatory stimuli on dental pulp stem cells (DPSCs) by assessing their proliferation and expression of genes as well as proteins in lipopolysaccharide (LPS)-induced microenvironment (iDPSCs). DPSCs were first characterized for their mesenchymal properties prior to challenging them with a series of LPS concentrations from 12 to 72 h. Following to this, their proliferation and inflammatory based genes as well as protein expression were assessed. iDPSCs had demonstrated significant expression of mesenchymal markers. Upon exposure to LPS, the viability dropped distinctly with increasing concentration, as compared to control (P 
  15. Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells
    Vasanthan P, Jayaraman P, Kunasekaran W, Lawrence A, Gnanasegaran N, Govindasamy V, et al.
    Naturwissenschaften, 2016 Aug;103(7-8):62.
    PMID: 27379400 DOI: 10.1007/s00114-016-1387-7
    Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.
  16. CRISPR/Cas9 in Stem Cell Research: Current Application and Future Perspective
    Patmanathan SN, Gnanasegaran N, Lim MN, Husaini R, Fakiruddin KS, Zakaria Z
    Curr Stem Cell Res Ther, 2018;13(8):632-644.
    PMID: 29895256 DOI: 10.2174/1574888X13666180613081443
    The clustered regularly interspaced short palindromic repeats-associated protein 9 or CRISPR/Cas9 system is one of the hottest topics discussed lately due to its robustness and effectiveness in genome editing. The technology has been widely used in life science research including microbial, plant, animal, and human cell studies. Combined with the pluripotency of stem cells, the technology represents a powerful tool to generate various cell types for disease modeling, drug screening, toxicology, and targeted therapies. Generally, the CRISPR/Cas9 system has been applied in genetic modification of pluripotent or multipotent stem cells, after which the cells are differentiated into specific cell types and used for functional analysis or even clinical transplantation. Recent advancement in CRISPR/Cas9 technology has widened the scope of stem cell research and its therapeutic application. This review provides an overview of the current application and the prospect of CRISPR/Cas9 technology, particularly in stem cell research and therapy.
  17. Effect of dental pulp stem cells in MPTP-induced old-aged mice model
    Gnanasegaran N, Govindasamy V, Simon C, Gan QF, Vincent-Chong VK, Mani V, et al.
    Eur J Clin Invest, 2017 Mar 30.
    PMID: 28369799 DOI: 10.1111/eci.12753
    BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic (DA-ergic) neurons in the substantia nigra (SN) and represented as a huge threat to the geriatric population. Cell replacement therapies (CRTs) have been proposed as a promising strategy to slow down or replace neuronal loss. Among the widely available cell sources, dental pulp stem cells (DPSCs) portray as an attractive source primarily due to their neural crest origin, ease of tissue procurement and less ethical hurdles.

    MATERIALS AND METHODS: We first demonstrated the in vitro differentiation ability of DPSCs towards DA-ergic-like cells before evaluating their neuro-protection/neuro-restoration capacities in MPTP-induced mice. Transplantation via intrathecal was performed with behavioural assessments being evaluated every fortnight. Subsequent analysis investigating their immuno-modulatory behaviour was conducted using neuronal and microglial cell lines.

    RESULTS: It was apparent that the behavioural parameters began to improve corresponding to tyrosine hydroxylase (TH), dopamine transporter (DAT) and dopamine decarboxylase (AADC) immunostaining in SN and striatum as early as 8-week post-transplantation (P < 0·05). About 60% restoration of DA-ergic neurons was observed at SN in MPTP-treated mice after 12-week post-transplantation. Similarly, their ability to reduce toxic effects of MPTP (DNA damages, reactive oxygen species and nitric oxide release) and regulate cytokine levels was distinctly noted (P < 0·05) upon exposure in in vitro model.

    CONCLUSIONS: Our results suggest that DPSCs may provide a therapeutic benefit in the old-aged PD mice model and may be explored in stem cell-based CRTs especially in geriatric population as an attempt towards 'personalized medicine'.

  18. Fulltext Quantification of mesenchymal stem cell growth rates through secretory and excretory biomolecules in conditioned media via Fresnel reflection
    Ahmad H, Thambiratnam K, Zulkifli AZ, Lawrence A, Jasim AA, Kunasekaran W, et al.
    Sensors (Basel), 2013 Sep 30;13(10):13276-88.
    PMID: 24084118 DOI: 10.3390/s131013276
    An efficient and low cost optical method for directly measuring the concentration of homogenous biological solutes is proposed and demonstrated. The proposed system operates by Fresnel reflection, with a flat-cleaved single-mode fiber serving as the sensor probe. A laser provides a 12.9 dBm sensor signal at 1,550 nm, while a computer-controlled optical power meter measures the power of the signal returned by the probe. Three different mesenchymal stem cell (MSC) lines were obtained, sub-cultured and trypsinized daily over 9 days. Counts were measured using a haemocytometer and the conditioned media (CM) was collected daily and stored at -80 °C. MSCs release excretory biomolecules proportional to their growth rate into the CM, which changes the refractive index of the latter. The sensor is capable of detecting changes in the number of stem cells via correlation to the change in the refractive index of the CM, with the measured power loss decreasing approximately 0.4 dB in the CM sample per average 1,000 cells in the MSC subculture. The proposed system is highly cost-effective, simple to deploy, operate, and maintain, is non-destructive, and allows reliable real-time measurement of various stem cell proliferation parameters.
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