Computer-Aided 2D and 3D quantification of human stem cell fate from in vitro samples using Volocity high performance image analysis software

Piltti, Katja; Haus, Daniel L.; Do, Eileen; Perez, H D; Anderson, Aileen J.; Cummings, Brian J.

doi:10.1016/j.scr.2011.05.005

Cited by 9 publications

(9 citation statements)

References 8 publications

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“…In comparison to rodent cells, hNSC require a longer time for in vitro differentiation. Our previous data suggest that a small proportion of hNSC remain uncommitted even after a 14 day in vitro differentiation period [15], however, evaluation of GFAP + cells in combination of stem cell markers Sox2 and Nestin indicates that by this timepoint GFAP expression correlates with astroglial lineage commitment rather than maintenance of neural progenitors [6]. After the last image acquisition at the 14 day timepoint, cells were fixed and triple-immunostained for lineage characterization using astroglial marker GFAP, oligodendroglial maker nuclear Olig2, or neuronal marker Map2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

Piltti

Cummings

Carta

et al. 2018

Methods

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Neural stem cell (NSC) cultures have been considered technically challenging for time-lapse analysis due to high motility, photosensitivity, and growth at confluent densities. We have tested feasibility of long-term live-cell time-lapse analysis for NSC migration and differentiation studies. Here, we describe a method to study the dynamics of cell cycle, migration, and lineage selection in cultured multipotent mouse or human NSCs using single-cell tracking during a long-term, 7–14 day live-cell time-lapse analysis. We used in-house made PDMS inserts with five microwells on a glass coverslip petri-dish to constrain NSC into the area of acquisition during long-term live-cell imaging. In parallel, we have defined image acquisition settings for single-cell tracking of cell cycle dynamics using Fucci-reporter mouse NSC for 7 days as well as lineage selection and migration using human NSC for 14 days. Overall, we show that adjustments of live-cell analysis settings can extend the time period of single-cell tracking in mouse or human NSC from 24–72 h up to 7–14 days and potentially longer. However, we emphasize that experimental use of repeated fluorescence imaging will require careful consideration of controls during acquisition and analysis.

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Section: Resultsmentioning

confidence: 99%

Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

Piltti

Cummings

Carta

et al. 2018

Methods

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“…Fluorescent images of immunostained slides were captured using random sampling on a FluoViewFV10i confocal microscope using a 320 objective, and saved as red, blue, and green channels. Images were merged and quantified using Volocity 5.0 software (28). For all experiments using hNSC, n = 8-10 images per condition per experiment and a minimum of three independent experiments was analyzed.…”

Section: Pmn Viability Analysis By Flow Cytometrymentioning

confidence: 99%

Neutrophils Induce Astroglial Differentiation and Migration of Human Neural Stem Cells via C1q and C3a Synthesis

Hooshmand

Nguyen

Piltti

et al. 2017

The Journal of Immunology

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Inflammatory processes play a key role in pathophysiology of many neurologic diseases/trauma, but the effect of immune cells and factors on neurotransplantation strategies remains unclear. We hypothesized that cellular and humoral components of innate immunity alter fate and migration of human neural stem cells (hNSC). In these experiments, conditioned media collected from polymorphonuclear leukocytes (PMN) selectively increased hNSC astrogliogenesis and promoted cell migration in vitro. PMN were shown to generate C1q and C3a; exposure of hNSC to PMN-synthesized concentrations of these complement proteins promoted astrogliogenesis and cell migration. Furthermore, in vitro, Abs directed against C1q and C3a reversed the fate and migration effects observed. In a proof-of-concept in vivo experiment, blockade of C1q and C3a transiently altered hNSC migration and reversed astroglial fate after spinal cord injury. Collectively, these data suggest that modulation of the innate/humoral inflammatory microenvironment may impact the potential of cell-based therapies for recovery and repair following CNS pathology.

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“…Briefly, prefiltered images were imported into Volocity and individual cells were found using intensity-based thresholding and then either excluded or retained based upon fluorescence and size. The software based object (cell) recognition was confirmed by visual observation of Volocity data images by an experienced user as detailed elsewhere (Piltti et al, 2011). Cells that (a) could not be tracked for the full time period, (b) were too close to each other, or (c) dividing were excluded from analysis.…”

Section: Tracking Of Cell Migrationmentioning

confidence: 99%

Region‐specific epithelial cell dynamics during branching morphogenesis

Hsu

Koo

Harunaga

et al. 2013

Developmental Dynamics

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Background Epithelial cells of developing embryonic organs, such as salivary glands, can display substantial motility during branching morphogenesis. Their dynamic movements and molecules involved in their migration are not fully characterized. Results We generated transgenic mice expressing photo-convertible KikGR and tracked the movements of individual cells highlighted by red fluorescence in different regions of developing salivary glands. Motility was highest for outer bud epithelial cells adjacent to the basement membrane, lower in inner bud cells, and lowest in duct cells. The highly motile outer cells contacting the basement membrane were pleomorphic, whereas inner cells were rounded. Peripheral cell motility was disrupted by antibodies inhibiting α6+β1 integrins and the non-muscle myosin II inhibitor blebbistatin. Inner bud cell migration was unaffected by these inhibitors, but their rate of migration was stimulated by inhibiting E-cadherin. Conclusions Cell motility in developing salivary glands was highest in cells in contact with the basement membrane. The basement membrane-associated motility of these outer bud cells depended on integrins and myosin II, but not E-cadherin. In contrast, motility of inner bud cells was restrained by E-cadherin. These findings identify the importance of integrin-dependent basement membrane association for the morphology, tissue organization, and lateral motility of morphogenetic epithelial cells.

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Computer-Aided 2D and 3D quantification of human stem cell fate from in vitro samples using Volocity high performance image analysis software

Cited by 9 publications

References 8 publications

Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

Neutrophils Induce Astroglial Differentiation and Migration of Human Neural Stem Cells via C1q and C3a Synthesis

Region‐specific epithelial cell dynamics during branching morphogenesis

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