Anirban Majumder

Neurotrophic Effects of Leukemia Inhibitory Factor on Neural Cells Derived from Human Embryonic Stem Cells

Various growth factor cocktails have been used to proliferate and then differentiate human neural progenitor (NP) cells derived from embryonic stem cells (ESC) for in vitro and in vivo studies. However, the cytokine leukemia inhibitory factor (LIF) has been largely overlooked. Here, we demonstrate that LIF significantly enhanced in vitro survival and promoted differentiation of human ESC‐derived NP cells. In NP cells, as well as NP‐derived neurons, LIF reduced caspase‐mediated apoptosis and reduced both spontaneous and H2O2‐induced reactive oxygen species in culture. In vitro, NP cell proliferation and the yield of differentiated neurons were significantly higher in the presence of LIF. In NP cells, LIF enhanced cMyc phosphorylation, commonly associated with self‐renewal/proliferation. Also, in differentiating NP cells LIF activated the phosphoinositide 3‐kinase and signal transducer and activator of transcription 3 pathways, associated with cell survival and reduced apoptosis. When differentiated in LIF+ media, neurite outgrowth and ERK1/2 phosphorylation were potentiated together with increased expression of gp130, a component of the LIF receptor complex. NP cells, pretreated in vitro with LIF, were effective in reducing infarct volume in a model of focal ischemic stroke but LIF did not lead to significantly improved initial NP cell survival over nontreated NP cells. Our results show that LIF signaling significantly promotes human NP cell proliferation, survival, and differentiation in vitro. Activated LIF signaling should be considered in cell culture expansion systems for future human NP cell‐based therapeutic transplant studies. STEM CELLS2012;30:2387–2399

Label-free characterization of emerging human neuronal networks.

The emergent self-organization of a neuronal network in a developing nervous system is the result of a remarkably orchestrated process involving a multitude of chemical, mechanical and electrical signals. Little is known about the dynamic behavior of a developing network (especially in a human model) primarily due to a lack of practical and non-invasive methods to measure and quantify the process. Here we demonstrate that by using a novel optical interferometric technique, we can non-invasively measure several fundamental properties of neural networks from the sub-cellular to the cell population level. We applied this method to quantify network formation in human stem cell derived neurons and show for the first time, correlations between trends in the growth, transport, and spatial organization of such a system. Quantifying the fundamental behavior of such cell lines without compromising their viability may provide an important new tool in future longitudinal studies.

Genetic manipulation of neural progenitors derived from human embryonic stem cells.

Human embryonic stem cell-derived neural progenitors (NP) present an important tool for understanding human development and disease. Optimal utilization of NP cells, however, requires an enhanced ability to monitor these cells in vitro and in vivo. Here we report production of the first genetically modified self-renewing human embryonic stem cell-derived NP cells that express fluorescent proteins under constitutive as well as lineage-specific promoters, enabling tracking and monitoring of cell fate. Nucleofection, transfection, and lentiviral transduction were compared for optimal gene delivery to NP cells. Transduction was most efficient in terms of transgene expression (37%), cell viability (39%), and long-term reporter expression (>3 months). Further, the constitutive gene promoters, cytomegalovirus, elongation factor 1alpha, and ubiquitin-C, exhibited comparable silencing (20-30%) in NP cells over a 2-month period, suggesting their suitability for long-term reporter expression studies. Transduced NP cells maintained their progenitor state and differentiation potential, as demonstrated by expression of endogenous NP markers and neuronal markers after differentiation. We also detected reporter expression in astrocytes generated from NP cells transduced with an astrocyte-specific gene promoter, glial fibrillary acidic protein, demonstrating the usefulness of this approach. The genetically manipulated NP cells described here offer great potential for live cell-tracking experiments, and a similar approach can as well be used for expression of proteins other than reporters.

Estimating weak ratiometric signals in imaging data. I. Dual-channel data

Ratiometric fluorescent indicators are becoming increasingly prevalent in many areas of biology. They are used for making quantitative measurements of intracellular free calcium both in vitro and in vivo, as well as measuring membrane potentials, pH, and other important physiological variables of interest to researchers in many subfields. Often, functional changes in the fluorescent yield of ratiometric indicators are small, and the signal-to-noise ratio (SNR) is of order unity or less. In particular, variability in the denominator of the ratio can lead to very poor ratio estimates. We present a statistical optimization method for objectively detecting and estimating ratiometric signals in dual-wavelength measurements of fluorescent, ratiometric indicators that improves on standard methods. With the use of an appropriate statistical model for ratiometric signals and by taking the pixel-pixel covariance of an imaging dataset into account, we are able to extract user-independent spatiotemporal information that retains high resolution in both space and time.

Inhibition of DNA methyltransferases and histone deacetylases induces astrocytic differentiation of neural progenitors.

Understanding how to specify rapid differentiation of human neural progenitor towards enriched non-transformed human astrocyte progenitors will provide a critical cell source to further our understanding of how astrocytes play a pivotal role in neural function and development. Human neural progenitors derived from pluripotent embryonic stem cells and propagated in adherent serum-free cultures provide a fate restricted renewable source for quick production of neural cells; however, such cells are highly refractive to astrocytogenesis and show a strong neurogenic bias, similar to neural progenitors from the early embryonic central nervous system (CNS). We found that several astrocytic genes are hypermethylated in such progenitors potentially preventing generation of astrocytes and leading to the proneuronal fate of these progenitors. However, epigenetic modification by Azacytidine (Aza-C) and Trichostatin A (TSA), with concomitant signaling from BMP2 and LIF in neural progenitor cultures shifts this bias, leading to expression of astrocytic markers as early as 5days of differentiation, with near complete suppression of neuronal differentiation. The resultant cells express major astrocytic markers, are amenable to co-culture with neurons, can be propagated as astrocyte progenitors and are cryopreservable. Although previous reports have generated astrocytes from pluripotent cells, the differentiation required extensive culture or selection based on cell surface antigens. The development of a label free and rapid differentiation process will expedite future derivation of astrocytes from various sources pluripotent cells including, but not limited to, human astrocytes associated with various neurological diseases.

New statistical methods enhance imaging of cameleon fluorescence resonance energy transfer in cultured zebrafish spinal neurons

Cameleons are genetically encoded fluorescence resonance energy transfer (FRET)-based Ca(2+) indicators. Attempts to use cameleons to detect neural activity in vertebrate systems have been largely frustrated by the small FRET signal, in contradistinction to the higher signals seen in Drosophila and Caenorhabditis elegans. We have developed a statistical optimization method capable of detecting small ratiometric signals in noisy imaging data, called statistical optimization for the analysis of ratiometric signals. Using this method, we can detect and estimate anticorrelated ratiometric signals with subcellular resolution in cultured, dissociated zebrafish spinal neurons expressing cameleon or loaded with fluo-4 and fura-red. This method may make it possible to use yellow cameleons for measuring neural activity at high resolution in transgenic animals.

Observational Study of Effects of Saroglitazar on Glycaemic and Lipid Parameters on Indian Patients with Type 2 Diabetes

Cardiovascular risk reduction is an important issue in the management of patients with Type 2 diabetes mellitus. Peroxisome proliferator activated receptor (PPAR) agonists favourably influence glycaemic and lipid parameters in patients with Type 2 diabetes and a dual PPAR agonist is expected to have favourable effect on both parameters. In this study we have analyzed the effect of Saroglitazar, a novel dual PPAR alpha & gamma agonist, on glycaemic and lipid parameters in Indian patients with Type 2 diabetes. After a mean follow-up period of 14 weeks in 34 patients, treatment with Saroglitazar, in a dose of 4 mg daily, resulted in significant improvement in both glycaemic and lipid parameters. There were significant mean reductions of fasting plasma glucose (36.71 mg/dl; p = 0.0007), post-prandial plasma glucose (66.29 mg/dl; p = 0.0005), glycosylated haemoglobin (1.13%; p < 0.0001), total cholesterol (48.16 mg/dl; p < 0.0001), low- density lipoprotein cholesterol (24.04 mg/dl; p = 0.0048), triglyceride (192.78 mg/dl; p = 0.0001), non-high density lipoprotein cholesterol (48.72 mg/dl; p < 0.0001) and the ratio of triglyceride and high density lipoprotein cholesterol (5.30; p = 0.0006). There was no significant change in body weight, blood pressure, high-density lipoprotein cholesterol and serum creatinine.

Nonviral Gene Delivery in Neural Progenitors Derived from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have been used to derive self-renewing neural progenitor (NP) cell lines. Here we describe methods to genetically modify these cells. Detailed methods for transfection and nucleofection in PSC-derived NP cells are presented. We have shown that nucleofection results in higher yield of GFP(+) NP cells as compared with transfection. However, nucleofection leads to higher cell death than transfection. Application of these methods allows for the development of novel tools to study human development and cellular differentiation. Genetically modified NPs have direct application in neural imaging, tracking neural cells, and for drug delivery to target organs using neural progenitor cells as carriers.

From the Cover: AstrocytesAre Protective Against Chlorpyrifos Developmental Neurotoxicity in Human Pluripotent Stem Cell-Derived Astrocyte-Neuron Cocultures

Human neural progenitor cells are capable of independent, directed differentiation into astrocytes, oligodendrocytes and neurons and thus offer a potential cell source for developmental neurotoxicity (DNT) systems. Human neural progenitor-derived astrocyte-neuron cocultured at defined ratios mimic cellular heterogeneity and interaction in the central nervous system. Cytochrome P450 enzymes are expressed at a relatively high level in astrocytes and may play a critical role in the biotransformation of endogenous or exogenous compounds, including chlorpyrifos, an organophosphate insecticide that affects the central nervous system. P450 enzymes metabolize chlorpyrifos to chlorpyrifos-oxon, which is then metabolized primarily to 3, 5, 6-trichloropyridinol in addition to diethylphosphate and diethylthiophosphate. These end metabolites are less neurotoxic than chlorpyrifos and chlorpyrifos-oxon. Our objective was to identify the interactive role of astrocytes and neurons in chlorpyrifos-induced human DNT. In neuron-only cultures, chlorpyrifos inhibited neurite length, neurite number and branch points per neuron in a dose-dependent manner during a 48 h exposure, starting at 10 μM. However, in astrocyte-neuron cocultures, astrocytes protected neurons from the effects of chlorpyrifos at higher concentrations, up to and including 30 μM chlorpyrifos and endogenous astrocyte P450 enzymes effectively metabolized chlorpyrifos. The P450 inhibitor SKF525A partly negated the protective effect of astrocytes, allowing reduction in branch points with chlorpyrifos (10 μM). Thus, the scalable and defined astrocyte-neuron cocultures model that we established here has potentially identified a role for P450 enzymes in astrocytic neuroprotection against chlorpyrifos and provides a novel model for addressing DNT in a more accurate multicellular environment.

Estimating weak ratiometric signals in imaging data. II. Meta-analysis with multiple, dual-channel datasets

Ratiometric fluorescent indicators are used for making quantitative measurements of a variety of physiological variables. Their utility is often limited by noise. This is the second in a series of papers describing statistical methods for denoising ratiometric data with the aim of obtaining improved quantitative estimates of variables of interest. Here, we outline a statistical optimization method that is designed for the analysis of ratiometric imaging data in which multiple measurements have been taken of systems responding to the same stimulation protocol. This method takes advantage of correlated information across multiple datasets for objectively detecting and estimating ratiometric signals. We demonstrate our method by showing results of its application on multiple, ratiometric calcium imaging experiments.