enumeration-of-neural-stem-cells-using-clonal-assays

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Agency for Science, Technology and Research (A*STAR)

Microscopy has become a de facto tool for biology. However, it suffers from a fundamental problem of poor contrast with increasing depth, as the illuminating light gets attenuated and scattered and hence can not penetrate through thick samples. The resulting decay of light intensity due to attenuation and scattering varies exponentially across the image. The classical space invariant deconvolution approaches alone are not suitable for the restoration of uneven illumination in microscopy images. In this paper, we present a novel physics-based field theoretical approach to solve the contrast degradation problem of light microscopy images. We have confirmed the effectiveness of our technique through simulations as well as through real field experimentations.

A field theoretical restoration method for images degraded by non-uniform light attenuation : an application for light microscopy.

Live imaging of neural stem cells and progenitors is important to follow the biology of these cells. Non-invasive imaging techniques, such as phase contrast microscopy, are preferred as neural stem cells are very sensitive to photoxic damage cause by excitation of fluorescent molecules. However, large illumination variations and weak foreground/background contrast make phase contrast images challenging for image processing. In the current work, we propose a new method to segment neurospheres imaged under phase contrast microscopy by employing high dynamic range imaging and advanced level-set method. The use of high dynamic range imaging enhances the fused image by expressing cell signatures from various exposure captures. We apply advanced level-set method in cell segmentation to improve the detection rate over simple methods such as thresholding. Validation experiments in the analysis of 21 images containing over 400 cells have demonstrated accuracy improvements over existing techniques.

Detection of unstained living neurospheres from phase contrast images with very large illumination variations.

Neural stem cells/neural progenitors (NSCs/NPs) are cells that give rise to the main cell types of the nervous system: oligodendrocytes, neurons, and astrocytes. Studying NSCs/NPs with time-lapse microscopy is critical to the understanding of the biology of these cells. However, NSCs/NPs are very sensitive to phototoxic damage, and therefore, fluorescent dyes cannot be used to follow these cells. Also, since in most of NSC/NP-related experiments, a large number of cells neesd to be monitored. Consequently, the acquisition of a huge amount of images is required. An additional difficulty is related to our original suspension living, tracking objective, behavior much closer to the natural, in vivo, way of development of the cells. Indeed, unlike adherent cells, suspension cells float freely in a liquid solution, thus, making their dynamics very different from that of adherent cells. As a result, existing visual tracking algorithms that have primarily been developed to track adherent cells are no longer adequate to tackle living cells in suspension. This paper presents a novel automated 3-D visual tracking of suspension living cells for time-lapse image acquisition using phase-contrast microscopy. This new tracking method can potentially strongly impact on current 3-D video microscopy methods, paving the way for innovative analysis of NSCs/NPs and as a result, on the study of neurodegenerative diseases.

Online 3-D tracking of suspension living cells imaged with phase-contrast microscopy.

Neurospheres are widely used to propagate and investigate neural stem cells (NSCs) and neural progenitors (NPs). However, the exact cell types present within neurospheres are still unknown. To identify cell types, we used single-cell mRNA profiling of 48 genes in 187 neurosphere cells. Using a clustering algorithm, we identified 3 discrete cell populations within neurospheres. One cell population [cluster unsorted (US) 1] expresses high Bmi1 and Hes5 and low Myc and Klf12. Cluster US2 shows intermediate expression of most of the genes analyzed. Cluster US3 expresses low Bmi1 and Hes5 and high Myc and Klf12. The mRNA profiles of these 3 cell populations correlate with a developmental timeline of early, intermediate, and late NPs, as seen in vivo from the mouse brain. We enriched the cell population for neurosphere-forming cells (NFCs) using morphological criteria of forward scatter (FSC) and side scatter (SSC). FSC/SSC(high) cells generated 2.29-fold more neurospheres than FSC/SSC(low) cells at clonal density. FSC/SSC(high) cells were enriched for NSCs and Lewis-X(+ve) cells, possessed higher phosphacan levels, and were of a larger cell size. Clustering of both FSC/SSC(high) and FSC/SSC(low) cells identified an NFC cluster. Significantly, the mRNA profile of the NFC cluster drew close resemblance to that of early NPs. Taken together, data suggest that the neurosphere culture system can be used to model central nervous system development, and that early NPs are the cell population that gives rise to neurospheres. In future work, it may be possible to further dissect the NFCs and reveal the molecular signature for NSCs.

Single-cell mRNA profiling identifies progenitor subclasses in neurospheres.

Neural stem and progenitor cells (NSCs and NPs) give rise to the central nervous system (CNS) during embryonic development. NSCs and NPs differentiate into three main cell-types of the CNS; astrocytes, oligodendrocytes, and neurons. NSCs are present in the adult CNS and are important in maintenance and repair. Adult NSCs hold great promise for endogenous or self-repair of the CNS. Intriguingly, NSCs have been implicated as the cells that give rise to brain tumors. Thus, the balance between survival, growth and differentiation is a critical aspect of NSC biology, during development, in the adult, and in disease processes. In this review, we survey what is known about survival factors that control both embryonic and adult NSCs. We discuss the neurosphere culture system as this is widely used to measure NSC activity and behavior in vitro and emphasize the importance of clonality. We define here NSC survival factors in their broadest sense to include any factor that influences survival and proliferation of NSCs and NPs. NSC survival factors identified to date include growth factors, morphogens, proteoglycans, cytokines, hormones, and neurotransmitters. Understanding NSC and NP interaction in response to these survival factors will provide insight to CNS development, disease and repair.

Neural stem cell survival factors.

We report here a novel fluorescent chemical probe which stains distinct neural stem/progenitor cells (NSPCs) by binding to acid ceramidase in mouse neurospheres. is distributed evenly or unevenly to the daughter cells during multiple mitoses enabling the live imaging of symmetric and asymmetric divisions of isolated NSPCs.

A fluorescent probe for imaging symmetric and asymmetric cell division in neurosphere formation.

Neural stem cells (NSCs) are isolated from primary brain tissue and propagated as a heterogeneous mix of cells, including neural progenitors. To date, NSCs have not been purified in vitro to allow study of their biology and utility in regenerative medicine. In this study, we identify C1qR1 as a novel marker for NSCs and show that it can be used along with Lewis-X (LeX) to yield a highly purified population of NSCs. Using time-lapse microscopy, we are able to follow NSCs forming neurospheres, allowing their visualization. Finally, using single-cell polymerase chain reaction (PCR), we determine the molecular signature of NSCs. The single-cell PCR data suggest that along with the Notch and Shh pathways, the Hippo pathway plays an important role in NSC activity.

Article	Year
A field theoretical restoration method for images degraded by non-uniform light attenuation : an application for light microscopy. Optics express\| PubMed ID: 19582043	2009
Detection of unstained living neurospheres from phase contrast images with very large illumination variations. Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference\| PubMed ID: 22255744	2011
Online 3-D tracking of suspension living cells imaged with phase-contrast microscopy. IEEE transactions on bio-medical engineering\| PubMed ID: 22510943	2012
Single-cell mRNA profiling identifies progenitor subclasses in neurospheres. Stem cells and development\| PubMed ID: 22834539	2012
Neural stem cell survival factors. Archives of biochemistry and biophysics\| PubMed ID: 23470250	2013
A fluorescent probe for imaging symmetric and asymmetric cell division in neurosphere formation. Chemical communications (Cambridge, England)\| PubMed ID: 24882713	2014
Purification, Visualization, and Molecular Signature of Neural Stem Cells. Stem cells and development\| PubMed ID: 26464067	2016

Shvetha Sankaran

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