Syed Ameer Basha Paspala

Current concept in neural regeneration research: NSCs isolation, characterization and transplantation in various neurodegenerative diseases and stroke: A review

Since last few years, an impressive amount of data has been generated regarding the basic in vitro and in vivo biology of neural stem cells (NSCs) and there is much far hope for the success in cell replacement therapies for several human neurodegenerative diseases and stroke. The discovery of adult neurogenesis (the endogenous production of new neurons) in the mammalian brain more than 40 years ago has resulted in a wealth of knowledge about stem cells biology in neuroscience research. Various studies have done in search of a suitable source for NSCs which could be used in animal models to understand the basic and transplantation biology before treating to human. The difficulties in isolating pure population of NSCs limit the study of neural stem behavior and factors that regulate them. Several studies on human fetal brain and spinal cord derived NSCs in animal models have shown some interesting results for cell replacement therapies in many neurodegenerative diseases and stroke models. Also the methods and conditions used for in vitro culture of these cells provide an important base for their applicability and specificity in a definite target of the disease. Various important developments and modifications have been made in stem cells research which is needed to be more specified and enrolment in clinical studies using advanced approaches. This review explains about the current perspectives and suitable sources for NSCs isolation, characterization, in vitro proliferation and their use in cell replacement therapies for the treatment of various neurodegenerative diseases and strokes.

Pluripotent stem cells - a review of the current status in neural regeneration.

Pharmacological or neurosurgical therapies currently in practice to treat the damage in various neurodegenerative disorders are not efficient in preventing progression or cure of these progressive neurodegenerative processes. Recently, a new approach, cell therapy using stem cell, is being evaluated. However, the use of this therapy in the treatment of these neurological diseases is highly restricted, mainly owing to several technical difficulties and limitations. The strategy of isolation and characterization of neural stem cells from various sources will probably provide a major impetus and open up an interesting, novel therapeutic modality for several neurodegenerative disorders. The high regenerative potential of damaged neural tissues suggests that various embryonic/adult sources serve as a proxy for neural stem cells for cell-based therapy.

Magnetic nanoparticle tagged stem cell transplantation in spinal cord injury: A promising approach for targeted homing of cells at the lesion site

Spinal cord injury (SCI) is a devastating condition that leads to significant morbidity. It results in a permanent neurological deficit due to damage of motor neurons. The resultant lesion is a barrier for “communication” between the brain and peripheral tissues, both at the effector as well as the receptor level. One of the primary goals of tissue engineering is to bridge the gap created by SCI and reestablish the damaged connections.[1,2] Stem cell transplantation has a great potential in designing effective therapies for SCI. Despite extensive therapeutic benefits, lack of a noninvasive and efficient cell delivery system, and poor engraftment limits the current role of stem cell therapies in SCI. Application of nanotechnology has already proven its potential in addressing some of these fundamental issues. The development of superparamagnetic iron oxide nanoparticles (SPIONPs) has provided a better pathway for the efficient delivery of stem cells at the target location.[3] A recent study by Tukmachev et al. has demonstrated the potential of SPIONPs in facilitating the homing of mesenchymal stem cells (MSCs) at the lesion site in a rat model of SCI.[4] The study demonstrated the accumulation of SPIO‐labeled MSCs in the vicinity of the lesion site from a distance of 10 cm from the site of injury by using an external magnetic system of 1.2T [Figure 1]. This study provides a better hope for future magnetic nanoparticle‐based delivery of stem cells at the desired site. However, few of the most important issues in cell‐based therapies for SCI are the formation of a glial scar and the regeneration of neurons and glia that undergo cell death soon after injury. Therefore, selection of the appropriate variety of stem cells has to be done for an effective treatment. In the same direction, our center has developed a novel gadolinium‐SPIO (Gd‐SPIO) magnetic nanoparticle by a soft chemical approach that provides a high biocompatibility. Gd‐SPIONPs produce enhanced sensitivity by decreasing the relaxation time of the proton longitudinally (T1) as well as transversely (T2). Thus, it may be a better choice for obtaining a contrast magnetic resonance imaging. The extremely low concentrations of the magnetic nanoparticles prevents the toxic side effects of free gadolinium ions from developing. Apart from its use in the generation of excellent contrast images, Gd‐SPIONPs have also shown a paramagnetic behavior due to their much greater proton relaxation per atom of iron than gadolinium. The paramagnetic activity of Gd‐SPIONPs in the presence of external magnetic fields is accompanied by its safe degradation and clearance from the biological system (unpublished data). Hence, directed transplantation of neuronal cells labeled with Gd‐SPIONPs may be considered as a better strategy for obtaining high contrast imaging and noninvasive cell trapping at the desired site. This combinational strategy of Gd‐SPIO labeled neuronal cells for the regeneration of chronically injured spinal cord would overcome the effects of the glial scar, inhibitory molecules, and help in utilizing tissue engineering strategies to bridge the lesion more effectively. Correspondence

Real-time cellular and molecular dynamics of bi-metallic self-therapeutic nanoparticle in cancer cells

Since last decades various kinds of nanoparticles have been functionalized to improve their biomedical applications. However, the biological effect of un-modified/non-functionalized bi-metallic magnetic nanoparticles remains under investigated. Herein we demonstrate a multifaceted non-functionalized bi-metallic inorganic Gd-SPIO nanoparticle which passes dual high MRI contrast and can kill the cancer cells through several mechanisms. The results of the present study demonstrate that Gd-SPIO nanoparticles have potential to induce cancer cell death by production of reactive oxygen species and apoptotic events. Furthermore, Gd-SPIO nanoparticles also enhance the expression levels of miRNA-199a and miRNA-181a-7p which results in decreased levels of cancer markers such as C-met, TGF-β and hURP. One very interesting finding of this study reveals side scatter-based real-time analysis of nanoparticle uptake in cancer cells using flow cytometry analysis. In conclusion, this study paves a way for future investigation of un-modified inorganic nanoparticles to purport enhanced therapeutic effect in combination with potential anti-tumor drugs/molecules in cancer cells.

Enhanced neuroprotective effect of mild-hypothermia with VPA against ethanol–mediated neuronal injury

INTRODUCTION Progress in understanding pathophysiological mechanisms and the development of targeted regenerative strategies have been hampered by the lack of predictive disease models, specifically for the conditions to which affected cell types are inaccessible. The present study has aimed to unearth the role of valproic acid (VPA) and mild hypothermia (MH) as promising strategy to enhance the neuroprotective mechanisms in undifferentiated and differentiated human neural precursor cells (hNPCs) against ethanol-induced damage. METHODS 5mM VPA alone or in combination with MH (33°C) was used to prevent the damage in proliferating and differentiating hNPCs. CD133+ve enriched hNPCs were cultured in vitro and exposed to 1M chronic ethanol concentration for 72h and followed by VPA and MH treatment for 24h. Morphometric analysis was performed to identify changes in neurospheres development and neuronal cell phenotypes. Flow cytometry and RT-qPCR analysis was performed to investigate alterations in key molecular pathways involved in cell survival and signaling. RESULTS Combination of VPA with MH displayed higher proportion of neuronal cell viability as compared to single treatment. Combination treatment was most effective in reducing apoptosis and reactive oxygen species levels in both the undifferentiated and differentiated hNPCs. VPA with MH significantly improved neuronal cell phenotype, active chromatin modeling, chaperon and multi-drug resistant pumps activity and expression of neuronal signaling molecules. CONCLUSION The study provided an efficient and disease specific in vitro model and demonstrated that combined treatment with VPA and MH activates several neuroprotective mechanisms and provides enhanced protection against ethanol-induced damage in cultured undifferentiated and differentiated hNPCs.

Role of drug transporters and heat shock proteins during ethanol exposure to human neural precursor cells and its lineages

INTRODUCTION Ethanol exposure to developing brain may alter the growth and differentiation of neurological cells resulting in unfavorable pathologies. Earlier studies have provided very limited mechanistic insights of cellular and molecular mechanisms which do not mimic with human situation due to varying cell types and poses potential challenges for investigation. Therefore, the present study was undertaken to evaluate the role of ABC transporters and heat shock proteins mediated response in human neural precursor cells (NPCs) and its lineages during proliferation and lineage differentiation against ethanol exposure. METHODS Effect of ethanol exposure was examined for neuronal cell survival and variation in cellular phenotype during neurospheres development and lineage differentiation. Generation of reactive oxygen species, and variation in cell cycle was identified along with transcriptional profiling for pluripotent markers (Nestin, NCAM, Sox-2, and Notch-2), drug transporters (ABCB1 and ABCG2) and stress protein (HSP70) during ethanol exposure. RESULTS ABC transporters as well as HSP70 mRNA expression was higher during proliferation as compared to differentiation with chronic ethanol (1 M) exposure (p < 0.01). Ethanol exposure resulted in higher variability in size and shape of developing neurospheres and decreased ability to form new neurosphere colonies. Significant changes were observed in dendrite development due to late ethanol exposure (p < 0.0001). CONCLUSION The present study demonstrated significant role of ABC transporters and HSP70 proteins in providing defense against ethanol-induced damage in human neurological cells. However, the over-expression of ABC transporter and HSP-70 proteins during such pathological conditions do not provide complete defense and additional strategies are required to repair the damage.

Molecular dynamics of pancreatic transcription factors in bioengineered humanized insulin producing neoorgan

BACKGROUND The present study has been aimed to identify molecular dynamics of pancreatic transcription factors (pTFs) during events of directed trans-differentiation of human hepatic progenitor cells (hHPCs) into insulin producing cells (InPCs) within bioengineered humanized neoorgan. The study demonstrates applicability of acellularized whole splenic scaffold (ASOS) to generate insulin producing humanized transplantable neoorgan through activation of pancreatic transcription factors. METHODS An efficient acellularization process was developed for xenogeneic rat spleen using change in different gradients of reagents perfusion through splenic artery for varying time points. The acellularized xenogeneic spleen scaffold was characterized thoroughly for preservation of extra-cellular matrix and retention of organ specific vasculature and mechanical properties. Further scaffolds were sterilized and repopulated with hHPCs which were triggered using a stage wise induction with growth factors and hyperglycemic challenge for trans-differentiation into InPCs. Dynamics of pTFs alone or simultaneously during induction process was identified using gene expression analysis and immunological staining. RESULTS The cells within the engineered neoorgan respond to growth factors and extrinsic hyperglycemic challenge and generate large number of InPCs under controlled dynamic regulation of pTFs. Highly controlled regulation of pTFs generates higher percentage of Nkx-6.1+/C-peptide+ cells within the engineered splenic scaffolds. Generation of high percentage of insulin and C-peptide positive cells in three-dimensional organ architecture responded better to hyperglycemic stimuli and produced higher quantity of insulin than 2D-culture system. CONCLUSION The present study provides a novel platform for designing effective regenerative strategies using whole organ scaffolds to control hyperglycemia under tight regulation of pTFs using humanized neoorgan system.

Bimetallic redox nanoprobe enhances the therapeutic efficacy of hyperthermia in drug-resistant cancer cells

Cancer nanotheranostics has emerged as one of the most promising fields of medicine wherein nano-sized molecules/agents are used for combined diagnosis and treatment of cancer. Despite promises of novel cancer therapeutic approaches, several crucial challenges have remained to be overcome for successful clinical translation of such agents. Hence, the present study has been aimed to investigate the therapeutic efficacy of bimetallic gadolinium super-paramagnetic iron oxide nanoformulation of ascorbic acid in synergism with hyperthermia on ascorbic acid-resistant breast cancer cells. This particular strategy provides real-time MRI-based non-invasive imaging of drug loading in resistant cancer cells along with highly enhanced therapeutic efficacy. This unique redox nanoprobe is capable of reversing drug-resistance mechanism in cancer cells and offers better therapeutic possibilities in targeted and effective destruction of drug-resistant cancer cells.

Pluripotent Stem Cells for Neural Regeneration

Neurodegenerative disorders remain challenging to treat using traditional pharmacological or neurosurgical approaches. In contrast, cell therapy is a promising strategy for ameliorating irreparable brain tissue damage during the process of neurogenesis. Currently, more efficient methodologies for isolating neural stem cells from a plethora of sources including hematopoietic stem cells and mesenchymal stem cells are continually being developed. The availability of neural stem cells would ensure that damaged neural tissues can be regenerated and fast-track translation from bed to bedside. In this chapter, we discuss various sources of neural stem cells, strategies for their isolation and characterization, and application of stem cells in the treatment of neurological disorders. Historically, clinical application of cell therapy for treating neurological diseases has been hindered due to numerous technical difficulties. Therefore, these barriers and potential ways of addressing them are also discussed.