Publications by authors named "Renjitha Gopurappilly"

14 Publications

  • Page 1 of 1

Pluripotent Stem Cell Derived Neurons as In Vitro Models for Studying Autosomal Recessive Parkinson's Disease (ARPD): PLA2G6 and Other Gene Loci.

Adv Exp Med Biol 2021 May 15. Epub 2021 May 15.

National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru, India.

Parkinson's disease (PD) is a neurodegenerative motor disorder which is largely sporadic; however, some familial forms have been identified. Genetic PD can be inherited by autosomal, dominant or recessive mutations. While the dominant mutations mirror the prototype of PD with adult-onset and L-dopa-responsive cases, autosomal recessive PD (ARPD) exhibit atypical phenotypes with additional clinical manifestations. Young-onset PD is also very common with mutations in recessive gene loci. The main genes associated with ARPD are Parkin, PINK1, DJ-1, ATP13A2, FBXO7 and PLA2G6. Calcium dyshomeostasis is a mainstay in all types of PD, be it genetic or sporadic. Intriguingly, calcium imbalances manifesting as altered Store-Operated Calcium Entry (SOCE) is suggested in PLA2G6-linked PARK 14 PD. The common pathways underlying ARPD pathology, including mitochondrial abnormalities and autophagic dysfunction, can be investigated ex vivo using induced pluripotent stem cell (iPSC) technology and are discussed here. PD pathophysiology is not faithfully replicated by animal models, and, therefore, nigral dopaminergic neurons generated from iPSC serve as improved human cellular models. With no cure to date and treatments aiming at symptomatic relief, these in vitro models derived through midbrain floor-plate induction provide a platform to understand the molecular and biochemical pathways underlying PD etiology in a patient-specific manner.
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http://dx.doi.org/10.1007/5584_2021_643DOI Listing
May 2021

SEPT7 regulates Ca entry through Orai channels in human neural progenitor cells and neurons.

Cell Calcium 2020 09 9;90:102252. Epub 2020 Jul 9.

National Centre for Biological Sciences (NCBS), Tata Institute of Fundamental Research, Bangalore, India. Electronic address:

Human neural progenitor cells (hNPCs) are self-renewing cells of neural lineage that can be differentiated into neurons of different subtypes. Here we show that SEPT7, a member of the family of filament-forming GTPases called septins, prevents constitutive Ca entry through the store-operated Ca entry channel, Orai in hNPCs and in differentiated neurons and is thus required for neuronal calcium homeostasis. Previous work in Drosophila neurons has shown that loss of one copy of the evolutionarily-conserved dSEPT7 gene leads to elevated Ca entry via Orai, in the absence of ER-Ca store depletion. We have identified an N-terminal polybasic region of SEPT7, known to interact with membrane-localized phospholipids, as essential for spontaneous calcium entry through Orai in hNPCs, whereas the GTPase domain of dSEPT7 is dispensable for this purpose. Re-organisation of Orai1 and the ER-Ca sensor STIM1 observed near the plasma membrane in SEPT7 KD hNPCs, supports the idea that Septin7 containing heteromers prevent Ca entry through a fraction of STIM-Orai complexes. Possible mechanisms by which SEPT7 reduction leads to opening of Orai channels in the absence of store-depletion are discussed.
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http://dx.doi.org/10.1016/j.ceca.2020.102252DOI Listing
September 2020

Measurement of Store-Operated Calcium Entry in Human Neural Cells: From Precursors to Differentiated Neurons.

Methods Mol Biol 2019 ;2029:257-271

National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India.

Calcium imaging in an ex-vivo setup is used to understand the calcium status of isolated cells or tissue. In this chapter we explain the use of the ratiometric chemical indicator Fura-2 which can be loaded into isolated cells in the form of lipophilic acetomethyl (AM) esters. Fura-2 is a combination of calcium chelator and fluorophore, and can be used with dual wavelength excitation (340/380 nm) for quantitative calcium concentrations. The cells can then be viewed using a fluorescence microscope and captured by a CCD camera. We specifically discuss the technique involved in understanding the endoplasmic reticulum (ER)-driven store-operated calcium entry (SOCE) in human neural precursors (NPCs) and spontaneously differentiated neurons derived from a pluripotent human embryonic stem cell (hESC) line. The derivation of neural precursors from stem cells and their subsequent spontaneous neural differentiation is also explained. The method can be used for various non-excitable and excitable cell types including neurons, be it freshly isolated, from frozen vials, or derived from different stem cell lines.
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http://dx.doi.org/10.1007/978-1-4939-9631-5_20DOI Listing
April 2020

Stable Knockdown in Self-Renewing Human Neural Precursors Promotes Premature Neural Differentiation.

Front Mol Neurosci 2018 11;11:178. Epub 2018 Jun 11.

National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru, India.

Ca signaling plays a significant role in the development of the vertebrate nervous system where it regulates neurite growth as well as synapse and neurotransmitter specification. Elucidating the role of Ca signaling in mammalian neuronal development has been largely restricted to either small animal models or primary cultures. Here we derived human neural precursor cells (NPCs) from human embryonic stem cells to understand the functional significance of a less understood arm of calcium signaling, Store-operated Ca entry or SOCE, in neuronal development. Human NPCs exhibited robust SOCE, which was significantly attenuated by expression of a stable shRNA-miR targeted toward the SOCE molecule, STIM1. Along with the plasma membrane channel Orai, STIM is an essential component of SOCE in many cell types, where it regulates gene expression. Therefore, we measured global gene expression in human NPCs with and without knockdown. Interestingly, pathways down-regulated through knockdown were related to cell proliferation and DNA replication processes, whereas post-synaptic signaling was identified as an up-regulated process. To understand the functional significance of these gene expression changes we measured the self-renewal capacity of NPCs with knockdown. The knockdown NPCs demonstrated significantly reduced neurosphere size and number as well as precocious spontaneous differentiation toward the neuronal lineage, as compared to control cells. These findings demonstrate that mediated SOCE in human NPCs regulates gene expression changes, that are likely to physiologically modulate the self-renewal and differentiation of NPCs.
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http://dx.doi.org/10.3389/fnmol.2018.00178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004407PMC
June 2018

Transcriptional profiling of human neural precursors post alcohol exposure reveals impaired neurogenesis via dysregulation of ERK signaling and miR-145.

J Neurochem 2017 Aug 21. Epub 2017 Aug 21.

School of Regenerative Medicine, Manipal University, Bangalore, India.

Gestational alcohol exposure causes a range of neuropsychological disorders by modulating neurodevelopmental genes and proteins. The extent of damage depends on the stage of the embryo as well as dosage, duration and frequency of exposure. Here, we investigated the neurotoxic effects of alcohol using human embryonic stem cells. Multiple read-outs were engaged to assess the proliferation and differentiation capacity of neural precursor cells upon exposure to 100 mM ethanol for 48 h corresponding to the blood alcohol levels for binge drinkers. Whole-genome analysis revealed a spatiotemporal dysregulation of neuronal- and glial-specific gene expression that play critical roles in central nervous system (CNS) development. Alterations observed in the transcriptome may be attributed to epigenetic constitution witnessed by differential histone H3 Lys-4/Lys-27 modifications and acetylation status. In-depth mRNA and protein expression studies revealed abrogated extracellular signal-regulated kinases signaling in alcohol-treated cells. Consistent with this finding, ingenuity pathway analysis and micro-RNA profiling demonstrated up-regulation of miR-145 by targeting the neural specifier Sox-2. We also show that the neurite branching complexity of tubulin, beta 3 class III+ neurons was greatly reduced in response to alcohol. Finally, in vivo studies using zebrafish embryos reconfirmed the in vitro findings. Employing molecular endpoints in a human model, this report indicates for the first time that acute alcohol exposure could lead to impaired brain development via perturbation of extracellular signal-regulated kinases pathway and miR-145. However, it still needs to be addressed whether these modulations sustain throughout development, compromising the ability of the individual during adulthood and aging.
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http://dx.doi.org/10.1111/jnc.14155DOI Listing
August 2017

Transcriptional profiling and functional network analyses of islet-like clusters (ILCs) generated from pancreatic stem cells in vitro.

Genomics 2015 Apr 23;105(4):211-9. Epub 2015 Jan 23.

School of Regenerative Medicine (SORM), Manipal University, Bangalore 560065, India. Electronic address:

We have earlier reported the generation of islet-like clusters (ILCs) from mesenchymal stromal cell (MSC)-like cells present in murine pancreas. Here we compare these ILCs to native primary islets by transcriptome screening. Genes were categorized into functional clusters and network analysis was done by Ingenuity Pathway Analysis (IPA). The fold changes for a selected panel of molecules were validated with quantitative real time PCR. A differential expression of 6516 genes (p-value ≤ 0.05, 1.5 fold change) with upregulated expression of numerous inflammatory and 'Epithelial to Mesenchymal Transition' molecules (EMT) was seen. A significant increase in the early β-cell marker expression in the ILCs indicated their progenitor status. Although not fully mature, ILCs offer certain advantages including the large number of easily inducible initiator MSCs. These 'naïve' cells may aid to devise protocols for generating functional islet equivalents. Moreover their maturation upon transplantation under local microenvironmental niche is highly possible.
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http://dx.doi.org/10.1016/j.ygeno.2015.01.003DOI Listing
April 2015

A Small Molecule Swertisin from Enicostemma littorale Differentiates NIH3T3 Cells into Islet-Like Clusters and Restores Normoglycemia upon Transplantation in Diabetic Balb/c Mice.

Evid Based Complement Alternat Med 2013 15;2013:280392. Epub 2013 Apr 15.

Department of Biochemistry, Faculty of Science, The M.S. University of Baroda, Vadodara Gujarat, 390 002, India.

Aim. Stem cell therapy is one of the upcoming therapies for the treatment of diabetes. Discovery of potent differentiating agents is a prerequisite for increasing islet mass. The present study is an attempt to screen the potential of novel small biomolecules for their differentiating property into pancreatic islet cells using NIH3T3, as representative of extra pancreatic stem cells/progenitors. Methods. To identify new agents that stimulate islet differentiation, we screened various compounds isolated from Enicostemma littorale using NIH3T3 cells and morphological changes were observed. Characterization was performed by semiquantitative RT-PCR, Q-PCR, immunocytochemistry, immunoblotting, and insulin secretion assay for functional response in newly generated islet-like cell clusters (ILCC). Reversal of hyperglycemia was monitored after transplanting ILCC in STZ-induced diabetic mice. Results. Among various compounds tested, swertisin, an isolated flavonoid, was the most effective in differentiating NIH3T3 into endocrine cells. Swertisin efficiently changed the morphology of NIH3T3 cells from fibroblastic to round aggregate cell cluster in huge numbers. Dithizone (DTZ) stain primarily confirmed differentiation and gene expression studies signified rapid onset of differentiation signaling cascade in swertisin-induced ILCC. Molecular imaging and immunoblotting further confirmed presence of islet specific proteins. Moreover, glucose induced insulin release (in vitro) and decreased fasting blood glucose (FBG) (in vivo) in transplanted diabetic BALB/c mice depicted functional maturity of ILCC. Insulin and glucagon expression in excised islet grafts illustrated survival and functional integrity. Conclusions. Rapid induction for islet differentiation by swertisin, a novel herbal biomolecule, provides low cost and readily available differentiating agent that can be translated as a therapeutic tool for effective treatment in diabetes.
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http://dx.doi.org/10.1155/2013/280392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639639PMC
May 2013

Pancreatic tissue resident mesenchymal stromal cell (MSC)-like cells as a source of in vitro islet neogenesis.

J Cell Biochem 2013 Oct;114(10):2240-7

Manipal Institute of Regenerative Medicine (MIRM), Manipal University, Bangalore, India.

Insufficient β-cell mass is a common denominator for both type 1 and type 2 diabetes. In vitro generation of β-cells from islet precursor cells, exocrine cells or ductal epithelia provide an alternative source of insulin-producing cells. However the presence of multipotent precursor cells within the pancreas is also deliberated. In this study we isolated mesenchymal stromal cell (MSC)-like cells from adult mouse pancreas by collagenase digestion. We used Knockout DMEM for our isolation procedure and the floating islets and acini were removed after 48 h. This strategy permitted the adhesion of stromal cells with typical mesenchymal morphology. These cells not only expressed MSC-specific markers like Sca-1, CD90.2, CD73, and CD44 but also generated osteocytes, adipocytes, and neurons when induced with specific growth media. Upon exposure to islet differentiation serum-free cocktail a significant upregulation of pancreatic markers like Nkx2.2, Nkx6.1, Pdx1, insulin, and somatostatin was seen. The differentiated islet-like cell aggregates (ICAs) secreted insulin which increased over the days in culture in presence of basal glucose levels. Taken together, our data strongly indicate that there is a tissue-resident precursor population within the pancreas that can be exploited for islet neogenesis in vitro.
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http://dx.doi.org/10.1002/jcb.24572DOI Listing
October 2013

Functional significance of GnRH and kisspeptin, and their cognate receptors in teleost reproduction.

Front Endocrinol (Lausanne) 2013 8;4:24. Epub 2013 Mar 8.

Brain Research Institute, School of Medicine and Health Sciences, Monash University Sunway Campus Selangor, Malaysia.

Guanine nucleotide binding protein (G-protein)-coupled receptors (GPCRs) are eukaryotic transmembrane proteins found in all living organisms. Their versatility and roles in several physiological processes make them the single largest family of drug targets. Comparative genomic studies using various model organisms have provided useful information about target receptors. The similarity of the genetic makeup of teleosts to that of humans and other vertebrates aligns with the study of GPCRs. Gonadotropin-releasing hormone (GnRH) represents a critical step in the reproductive process through its cognate GnRH receptors (GnRHRs). Kisspeptin (Kiss1) and its cognate GPCR, GPR54 (=kisspeptin receptor, Kiss-R), have recently been identified as a critical signaling system in the control of reproduction. The Kiss1/Kiss-R system regulates GnRH release, which is vital to pubertal development and vertebrate reproduction. This review highlights the physiological role of kisspeptin-Kiss-R signaling in the reproductive neuroendocrine axis in teleosts through the modulation of GnRH release. Moreover, we also review the recent developments in GnRHR and Kiss-R with respect to their structural variants, signaling mechanisms, ligand interactions, and functional significance. Finally, we discuss the recent progress in identifying many teleost GnRH-GnRHR and kisspeptin-Kiss-R systems and consider their physiological significance in the control of reproduction.
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http://dx.doi.org/10.3389/fendo.2013.00024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591744PMC
March 2013

Embryonic fibroblasts represent a connecting link between mesenchymal and embryonic stem cells.

Dev Growth Differ 2013 Apr 27;55(3):330-40. Epub 2013 Feb 27.

Manipal Institute of Regenerative Medicine, Manipal University Branch Campus, Yelahanka, Bangalore, 560 065, Karnataka, India.

It is well established that fibroblasts and mesenchymal stem cells (MSC) share several characteristics with subtle differences. However, no study highlighting the versatility of fibroblasts beyond their multipotentiality has been reported so far. Mouse embryonic fibroblasts (MEFs) are widely used as feeder layers to support the growth of embryonic stem cells (ESC). We hypothesized that MEF may retain ES-like features in concurrence to their developmental hierarchy in addition to their multipotent nature. Hence, we performed a comparative assessment of MEF and ESC to determine their ability to differentiate into cell types other than mesoderm as well as capacity to form teratoma using routine in vitro and in vivo techniques. MEF were derived by trypsin/ EDTA (ethylenediaminetetraacetic acid) digestion from E13.5 embryos after removing heads and viscera following plastic adherence. MEFs robustly proliferated in culture until passage 15 and formed aggregates by hanging drop method. Flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry revealed the presence of key MSC markers such as CD90, CD73, Sca-1, CD44, CD29, Vimentin and absence of CD45. Additionally, they expressed SSEA-1, Oct-4, Nanog, Sox-2 and ABCG2 as pluripotency markers; Nestin, β-III tubulin, Otx-2 (ectoderm); MEF-2, Mesp2, GATA-2 (mesoderm) and GATA-4, α-amylase, PDX-1 (endoderm) as tri-lineage markers. Furthermore, MEFs formed representative tissues from all three germ layers upon transplantation into Balb/c mice. These unique abilities of MEF to exhibit pluripotency, in addition to fibroblast characteristics and their ready availability with less ethical concerns and low maintenance requirements make them an attractive model for further exploration as a possible tool for regenerative medicine.
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http://dx.doi.org/10.1111/dgd.12043DOI Listing
April 2013

Can multiple intramuscular injections of mesenchymal stromal cells overcome insulin resistance offering an alternative mode of cell therapy for type 2 diabetes?

Med Hypotheses 2012 Mar 20;78(3):393-5. Epub 2011 Dec 20.

Manipal Institute of Regenerative Medicine, Manipal University, Bangalore 560 071, India.

Insulin resistance is a hallmark of type 2 diabetes (T2D). The mechanisms underpinning β-cell mass expansion and their functionality in insulin-resistant states still remain elusive. It has recently been shown that insulin resistance in skeletal muscles leads to production of myokines that impact negatively on β-cell function. We hypothesize that multiple intramuscular injections (IM) of mesenchymal stromal cells (MSCs) at different sites would aid in countering the insulin resistance in T2D. These IM injections are expected to have dual effects in overcoming muscle insulin resistance. It is likely to modulate the micro environmental niche of insulin-insensitive myocytes under the influence of paracrine secretions from MSCs, in turn changing the myokine secretion pattern to positively regulate β-cell function. Further, it may stimulate the satellite cell population to generate new myocytes, which would be insulin-sensitive. If our hypothesis proves to be right, it might offer a user-friendly approach to control T2D.
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http://dx.doi.org/10.1016/j.mehy.2011.11.021DOI Listing
March 2012

Pancreatic progenitors: The shortest route to restore islet cell mass.

Islets 2011 Nov-Dec;3(6):295-301. Epub 2011 Nov 1.

Department of Biochemistry/Stem Cell Research, National Institute of Nutrition, Hyderabad, India.

The regenerative process of the pancreas is of interest because the main pathogenesis of diabetes mellitus is an inadequate number of insulin-producing β-cells. The functional mass of β-cells is decreased in most forms of diabetes, so replacing missing β-cells or triggering their regeneration may allow for improved diabetes treatment. Therefore, expansion of the β-cell mass from endogenous sources, either in vivo or in vitro, represents an area of increasing interest. The mechanism of islet regeneration remains poorly understood, but the identification of islet progenitor sources is critical for understanding β-cell regeneration. One potential source is the islet proper, via the de-differentiation, proliferation and redifferentiation of facultative progenitors residing within the islet. The new pancreatic islets derived from progenitor cells present within the ducts have been reported, but the existence and identity of the progenitor cells have been debated. In this mini-review, we focus primarily on pancreatic progenitors, which are islet progenitors capable of differentiating into insulin producing cells. We also emphasize the importance of pancreatic progenitors as a target for stem cell therapy for diabetes.
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http://dx.doi.org/10.4161/isl.3.6.17704DOI Listing
June 2012

Stem cells in stroke repair: current success and future prospects.

CNS Neurol Disord Drug Targets 2011 Sep;10(6):741-56

Brain Research Institute at Monash Sunway, School of Medicine and Health Sciences, Monash University, Selangor, Malaysia.

Stroke causes a devastating insult to the brain resulting in severe neurological deficits because of a massive loss of different neurons and glia. In the United States, stroke is the third leading cause of death. Stroke remains a significant clinical unmet condition, with only 3% of the ischemic patient population benefiting from current treatment modalities, such as the use of thrombolytic agents, which are often limited by a narrow therapeutic time window. However, regeneration of the brain after ischemic damage is still active days and even weeks after stroke occurs, which might provide a second window for treatment. Neurorestorative processes like neurogenesis, angiogenesis and synaptic plasticity lead to functional improvement after stroke. Stem cells derived from various tissues have the potential to perform all of the aforementioned processes, thus facilitating functional recovery. Indeed, transplantation of stem cells or their derivatives in animal models of cerebral ischemia can improve function by replacing the lost neurons and glial cells and by mediating remyelination, and modulation of inflammation as confirmed by various studies worldwide. While initially stem cells seemed to work by a 'cell replacement' mechanism, recent research suggests that cell therapy works mostly by providing trophic support to the injured tissue and brain, fostering both neurogenesis and angiogenesis. Moreover, ongoing human trials have encouraged hopes for this new method of restorative therapy after stroke. This review describes up-to-date progress in cell-based therapy for the treatment of stroke. Further, as we discuss here, significant hurdles remain to be addressed before these findings can be responsibly translated to novel therapies. In particular, we need a better understanding of the mechanisms of action of stem cells after transplantation, the therapeutic time window for cell transplantation, the optimal route of cell delivery to the ischemic brain, the most suitable cell types and sources and learn how to control stem cell proliferation, survival, migration, and differentiation in the pathological environment. An integrated approach of cell-based therapy with early-phase clinical trials and continued preclinical work with focus on mechanisms of action is needed.
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http://dx.doi.org/10.2174/187152711797247894DOI Listing
September 2011

Current status and prospective application of stem cell-based therapies for spinal cord injury.

Curr Stem Cell Res Ther 2011 Jun;6(2):93-104

Stempeutics Research Malaysia Sdn. Bhd., Technology Park Malaysia, Kuala Lumpur 57000, Malaysia.

Spinal cord injuries (SCIs) are a common form of trauma that leaves a huge trail of morbidity and human suffering in its wake. They occur mostly among the young, causing severe physical, psychological, social and economic burdens. The treatment of this condition has rather been disappointing; most of the management strategies being mainly supportive and prophylactic. In recent years there has been an emerging interest in the use of stem cells to regenerate the nervous tissue that has been damaged or lost. Although there has been much hype and unfounded hope, modest successes have been witnessed, and it is possible that these therapeutic strategies may have much more to offer in the future. This paper will review the current strategies of exploring cell-based therapies, mainly different types of stem cells to treat SCI along with the evidence that has been accumulated over the past decade in a rational bench-to-bedside approach. Furthermore, critical aspects such as the mode of delivery and ethical considerations are also discussed along with feasible suggestions for future translational research to provide a contextual picture of the current state of advancements in this field. The impediments to regeneration in the site of injury are briefly explained along with the benefits and drawbacks of different cell types used in the treatment of this condition. We hope that this review will offer a significant insight into this challenging clinical condition.
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http://dx.doi.org/10.2174/157488811795495477DOI Listing
June 2011