Gurudutta Gangenahalli

Stromal-derived factor-1/CXCR4 signaling: indispensable role in homing and engraftment of hematopoietic stem cells in bone marrow.

Homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) in bone marrow is the major determining factor in success of hematopoietic stem cell transplantation. This is a complex, multistep process orchestrated by the coordinated interplay between adhesion molecules, cytokines, growth factors, and regulatory cofactors, many of which remain to be defined. Recent studies have highlighted the pivotal role of unique stromal-derived factor-1 (SDF-1)/CXCR4 signaling in the regulation of HSPC homing and subsequent engraftment. In addition, studies suggest that SDF-1/CXCR4 signaling acts as an essential survival-promoting factor of transplanted HSPCs as well as maintenance of quiescent HSCs in bone marrow niche. These pleiotropic effects exerted by SDF-1/CXCR4 axis make this unique signaling initiator very promising, not only for optimal hematopoietic reconstitution but also for the development of innovative approaches to achieve restoration, regeneration, or repair of other damaged tissues potentially amendable to reversal by stem cell transplantation. This goal can only be achieved when the role of SDF-1/CXCR4 axis in hematopoietic transplantation is clearly defined. Hence, this review presents current knowledge of the mechanisms through which SDF-1/CXCR4 signaling promotes restoration of hematopoiesis by regulating the homing and engraftment of HSPCs.

Induced Pluripotent Stem Cells: Fundamentals and Applications of the Reprogramming Process and its Ramifications on Regenerative Medicine

ObjectiveTo provide a comprehensive source of information about the reprogramming process and induced pluripotency.BackgroundThe ability of stem cells to renew their own population and to differentiate into specialized cell types has always attracted researchers looking to exploit this potential for cellular replacement therapies, pharmaceutical testing and studying developmental pathways. While adult stem cell therapy has already been brought to the clinic, embryonic stem cell research has been beset with legal and ethical impediments.FocusThe conversion of human somatic cells to human induced pluripotent stem cells (hiPSCs), which are equivalent to human embryonic stem cells (hESCs), provides a system to sidestep these barriers and expedite pluripotent stem cell research for the aforementioned purposes. However, being a very recent discovery, iPSCs have yet to overcome many other obstacles and criticism to be proven safe and feasible for clinical use.MethodologyThis review introduces iPSC, the various methods that have been used to generate them and their pros and cons. It also covers in detail the pluripotency factors responsible for iPSC generation as well as the signaling pathways, epigenetic modifications and miRNA regulation implicated in the reprogramming process. The known molecular crosstalk between these reprogramming regulators is also illuminated. We will also mention the molecular compounds which have been shown to either replace one or more genetic factors or improve overall efficiency and kinetics of iPSC induction.ConclusionTo conclude, we will briefly discuss the current problems that hinder bench to bedside translation of iPSC research as well as the possible steps that can bring iPSC therapy and other potential applications closer to fruition.

High Throughput Transcriptome Profiling of Lithium Stimulated Human Mesenchymal Stem Cells Reveals Priming towards Osteoblastic Lineage

Human mesenchymal stem cells (hMSCs) present in the bone marrow are the precursors of osteoblasts, chondrocytes and adipocytes, and hold tremendous potential for osteoregenerative therapy. However, achieving directed differentiation into osteoblasts has been a major concern. The use of lithium for enhancing osteogenic differentiation has been documented in animal models but its effect in humans is not clear. We, therefore, performed high throughput transcriptome analysis of lithium-treated hMSCs to identify altered gene expression and its relevance to osteogenic differentiation. Our results show suppression of proliferation and enhancement of alkaline phosphatase (ALP) activity upon lithium treatment of hMSCs under non-osteogenic conditions. Microarray profiling of lithium-stimulated hMSC revealed decreased expression of adipogenic genes (CEBPA, CMKLR1, HSD11B1) and genes involved in lipid biosynthesis. Interestingly, osteoclastogenic factors and immune responsive genes (IL7, IL8, CXCL1, CXCL12, CCL20) were also downregulated. Negative transcriptional regulators of the osteogenic program (TWIST1 and PBX1) were suppressed while genes involved in mineralization like CLEC3B and ATF4 were induced. Gene ontology analysis revealed enrichment of upregulated genes related to mesenchymal cell differentiation and signal transduction. Lithium priming led to enhanced collagen 1 synthesis and osteogenic induction of lithium pretreated MSCs resulted in enhanced expression of Runx2, ALP and bone sialoprotein. However, siRNA-mediated knockdown of RRAD, CLEC3B and ATF4 attenuated lithium-induced osteogenic priming, identifying a role for RRAD, a member of small GTP binding protein family, in osteoblast differentiation. In conclusion, our data highlight the transcriptome reprogramming potential of lithium resulting in higher propensity of lithium “primed” MSCs for osteoblastic differentiation.

Cell death regulation by B-cell lymphoma protein

Bcl-2 (B Cell Lymphoma) protein is an anti-apoptotic member of Bcl-2 family, which is comprised of pro- and anti-apoptotic members. It regulates cellular proliferation and death by inter- and intra-family interactions. It has a potential to suppress apoptotic cell death under variety of stress conditions by modulating mitochondrial transmembrane potential. However, prevalence of constitutively activated Bcl-2 cellular activity is not always required in cells; a mechanism likely exists in cells, which controls its activity. When expression of Bcl-2 is unregulated, it generates lymphoma like, follicular B-cell lymphoma. This article reviews the structural and functional regulation of Bcl-2 activity at transcriptional, translational, domain, structural and post-translational level, which also accounts for the effects of its deletion and site-directed mutants in the regulation of cellular proliferation and differentiation in vitro and in vivo. This concisely reviewed information on Bcl-2 helps us to update our understanding of cell death and its modulation by Bcl-2 and its mutant’s interaction, which has gained therapeutic benefits in cell growth and proliferation, particularly for sensitive human hematopoietic stem cells.

Early monitoring and quantitative evaluation of macrophage infiltration after experimental traumatic brain injury: A magnetic resonance imaging and flow cytometric analysis

ABSTRACT The inflammatory response following traumatic brain injury (TBI) is regulated by phagocytic cells. These cells comprising resident microglia and infiltrating macrophages play a pivotal role in the interface between early detrimental and delayed beneficial effects of inflammation. The aim of the present study was to monitor the early effect of monocyte/phagocytic accumulation and further to explore its kinetics in TBI mice. Localized macrophage population was monitored using ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle enhanced in vivo serial magnetic resonance imaging (MRI). Flow cytometry based gating study was performed to discriminate between resident microglia (Ly6G− CD11b+ CD45low) and infiltrating macrophages (Ly6G− CD11b+ CD45high) at the injury site. The T2* relaxation analysis revealed that maximum macrophage infiltration occurs between 66 and 72 h post injury (42–48 h post administration of USPIO) at the site of inflammation. This imaging data was well supported by iron oxide specific Prussian blue staining and macrophage specific F4/80 immunohistochemistry (IHC) analysis. Quantitative real‐time PCR analysis found significant expression of monocyte chemoattractant protein‐1 (MCP‐1) at 72 h post injury. Also, we found that flow cytometric analysis demonstrated a 7‐fold increase in infiltrating macrophages around 72 h post injuries as compared to control. The MR imaging in combination with flow cytometric analysis enabled the dynamic measurement of macrophage infiltration at the injury site. This study may help in setting an optimal time window to intervene and prevent damage due to inflammation and to increase the therapeutic efficacy. Graphical abstract Figure. No caption available. HighlightsMacrophages were labeled in vivo via intravenous injection of USPIO in TBI mice.Maximum macrophage infiltration occurred between 66 and 72 h post injuries in MRI study.A 7‐fold increase in infiltrating macrophages occurred after 72 h post injury.PB staining and F4/80 staining were well supported with quantitative T2* data.

Molecular dynamics simulations of the Bcl-2 protein to predict the structure of its unordered flexible loop domain

B-cell lymphoma (Bcl-2) protein is an anti-apoptotic member of the Bcl-2 family. It is functionally demarcated into four Bcl-2 homology (BH) domains: BH1, BH2, BH3, BH4, one flexible loop domain (FLD), a transmembrane domain (TM), and an X domain. Bcl-2’s BH domains have clearly been elucidated from a structural perspective, whereas the conformation of FLD has not yet been predicted, despite its important role in regulating apoptosis through its interactions with JNK-1, PKC, PP2A phosphatase, caspase 3, MAP kinase, ubiquitin, PS1, and FKBP38. Many important residues that regulate Bcl-2 anti-apoptotic activity are present in this domain, for example Asp34, Thr56, Thr69, Ser70, Thr74, and Ser87. The structural elucidation of the FLD would likely help in attempts to accurately predict the effect of mutating these residues on the overall structure of the protein and the interactions of other proteins in this domain. Therefore, we have generated an increased quality model of the Bcl-2 protein including the FLD through modeling. Further, molecular dynamics (MD) simulations were used for FLD optimization, to predict the flexibility, and to determine the stability of the folded FLD. In addition, essential dynamics (ED) was used to predict the collective motions and the essential subspace relevant to Bcl-2 protein function. The predicted average structure and ensemble of MD-simulated structures were submitted to the Protein Model Database (PMDB), and the Bcl-2 structures obtained exhibited enhanced quality. This study should help to elucidate the structural basis for Bcl-2 anti-apoptotic activity regulation through its binding to other proteins via the FLD.

Increased transverse relaxivity in ultrasmall superparamagnetic iron oxide nanoparticles used as MRI contrast agent for biomedical imaging.

Synthesis of a contrast agent for biomedical imaging is of great interest where magnetic nanoparticles are concerned, because of the strong influence of particle size on transverse relaxivity. In the present study, biocompatible magnetic iron oxide nanoparticles were synthesized by co-precipitation of Fe2+ and Fe3+ salts, followed by surface adsorption with reduced dextran. The synthesized nanoparticles were spherical in shape, and 12 ± 2 nm in size as measured using transmission electron microscopy; this was corroborated with results from X-ray diffraction and dynamic light scattering studies. The nanoparticles exhibited superparamagnetic behavior, superior T2 relaxation rate and high relaxivities (r1  = 18.4 ± 0.3, r2  = 90.5 ± 0.8 s-1 mM-1 , at 7 T). MR image analysis of animals before and after magnetic nanoparticle administration revealed that the signal intensity of tumor imaging, specific organ imaging and whole body imaging can be clearly distinguished, due to the strong relaxation properties of these nanoparticles. Very low concentrations (3.0 mg Fe/kg body weight) of iron oxides are sufficient for early detection of tumors, and also have a clear distinction in pre- and post-enhancement of contrast in organs and body imaging. Many investigators have demonstrated high relaxivities of magnetic nanoparticles at superparamagnetic iron oxide level above 50 nm, but this investigation presents a satisfactory, ultrasmall, superparamagnetic and high transverse relaxivity negative contrast agent for diagnosis in pre-clinical studies. Copyright

Potential stem cell labeling ability of poly-L-lysine complexed to ultrasmall iron oxide contrast agent: An optimization and relaxometry study

For non-invasive stem cells tracking through MRI, it is important to understand the efficiency of in vitro labeling of stem cells with iron oxide with regard to its relaxation behavior. In this study, we have carried out a pilot study of labeling mice mesenchymal stem cells (mMSCs) with ultrasmall superparamagnetic iron oxide (USPIO) entrapped with poly-L-lysine (PLL) in different ratios and incubated with different times. Our results demonstrated that 50:1.5 µg/ml of iron oxide and PLL at an incubation time of 6h with 10% serum concentration are sufficient enough for effective labeling. Optimized labeling showed that >98% of viability and <3% toxicity were observed at a total iron content of 11.8 pg/cell. In vitro relaxometry study showed that almost a 6.6 fold reduction in transverse relaxation time (T2) was observed after labeling as compared to unlabeled. IO-PLL complex was more effective than iron oxide alone in labeling and a detectable lower limit found to be hundred with optimized concentration. Significant increase in Oct-4 expression on day-3 after labeling was observed, whereas CD146 expression remains unchanged in real time RT-PCR. This optimized labeling method of MSCs may be very useful for cellular MRI and stem cells tracking studies.

Therapeutic Prospective of Infused Allogenic Cultured Mesenchymal Stem Cells in Traumatic Brain Injury Mice: A Longitudinal Proton Magnetic Resonance Spectroscopy Assessment

Improved therapeutic assessment of experimental traumatic brain injury (TBI), using mesenchymal stem cells (MSCs), would immensely benefit its therapeutic management. Neurometabolite patterns at injury site, measured with proton magnetic resonance spectroscopy (1H‐MRS) after MSCs transplantation, may serve as a bio‐indicator of the recovery mechanism. This study used in vivo magnetic resonance imaging and 1H‐MRS to evaluate the therapeutic prospects of implanted MSCs at injury site in experimental mice longitudinally up to 21 days. Negative tissue contrast and cytotoxic edema formation were observed in susceptibility‐based contrast (T2*) and an apparent diffusion coefficient map, respectively. Lesion site showed decreased N‐acetylaspartate, total choline, myo‐inositol, total creatine, glutamate‐glutamine complex, and taurine neurometabolic concentrations by 1H‐MRS investigation. There was a considerable decrease in locomotor activity, depression index, and cognitive index after TBI. It may, therefore, be inferred that MSC transplantation prompted recovery by decreasing negative signals and edema, restoring metabolites to baseline concentrations, and enhancing behavioral activity. Overall findings support the potential of MSC transplantation for the enhancement of endogenous neuroprotective responses, which may provide future clinical applications for translating laboratory research into therapeutic clinical advances. Stem Cells Translational Medicine 2017;6:316–329

Transgene expression study of CXCR4 active mutants. Potential prospects in up-modulation of homing and engraftment efficiency of hematopoietic stem/progenitor cells.

Homing and engraftment, a determining factor in hematopoietic stem cell transplantation success is defined as a process through which hematopoietic stem/progenitor cells (HSPCs) lodge recipient bone marrow. SDF-1/CXCR4 axis acts as a principle regulator in homing and engraftment, however, CXCR4 signaling is dependent upon expression of CXCR4 and its ligand SDF-1, which is highly dynamic. Hence, present investigation was aimed to explore the potential of CXCR4 constitutive active mutants (CXCR4-CAMs) in overcoming the limitation of CXCR4 signaling and up-modulate its efficiency in homing and engraftment. Regulated transgene expression study of these mutants revealed their significantly enhanced cell adhesion efficiency to endothelium and extracellular matrix protein. This altogether indicates promising prospects of CXCR4-CAMs in research aimed to improve HSPCs engraftment efficiency.