Clonal Neural Stem Cells from Human Embryonic Stem Cell Colonies

Chaddah, Radha; Arntfield, Margot; Runciman, Susan; Clarke, Laura; Kooy, Derek van der

doi:10.1523/jneurosci.3286-11.2012

Cited by 42 publications

(35 citation statements)

References 53 publications

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“…In a single cell-labeling experiment and live image capture, we found that red-labeled porcine germ cells clustered together to form colonies when red-labeled GFRa-1-positive germ cells were added to the total testicular cell culture. A similar finding has been reported in the colony formation experiment; a neural stem cells colony, which was differentiated from hESCs, was formed by aggregation of red and green hESCs that were cultured in the same well (Chaddah et al, 2012). These results suggest that aggregation of single germ cells, which have germ and stem cell characteristics, occurs in GDC formation in a manner similar to that observed in hESC colony formation.…”

Section: Discussionsupporting

confidence: 87%

In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells

Lee

Tae

et al. 2016

Cellular Reprogramming

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Embryonic body-like colony formation is a unique pattern in male germ cell cultures, including spermatogonial stem cells. However, detailed information of the colony formation has not yet been sufficiently reported in male germ cell culture. To elucidate the formation of germ cell-derived colony (GDC), glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1)-positive pig germ cells were isolated using an immunomagnetic cell isolation method and labeled with red- or green-fluorescent dye. In GDC culture, red-fluorescent-labeled germ cells were evenly distributed in the wells from day 1 to 4, and they clustered together at the time of GDC formation on day 6. Interestingly, feeder cells migrated to the site of colony formation as spermatogonia carriers. Furthermore, when freshly prepared green-labeled GFRα-1-positive germ cells were added, mixed-fluorescent dye (red and green) colonies were observed. On bromodeoxyuridine (BrdU) treatment, 58% ± 3.13% of germ cells were positive to protein gene product 9.5 but negative to BrdU cells. Immunocytochemistry and reverse transcription-polymerase chain reaction results showed that cultured GDC cells were positive to stem cell- and pig germ cell-specific marker genes. In conclusion, in vitro formation of GDCs is mainly dependent on the aggregation of single germ cells as well as on the slow proliferation of germ cells.

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Section: Discussionsupporting

confidence: 87%

In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells

Lee

Tae

et al. 2016

Cellular Reprogramming

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“…Since it is hard to control precise differentiation due to the heterogeneity of cells in high-density culture, lots of effort has been made to culture NSCs at low density. For clonal culture, cells at 10 cells μl −1 or less are plated for neurosphere culture 51–53 . However, van der Kooy and his colleagues reported that even looking at neurosphere cultures at clonal density during experiments can result in chimera-sphere-formation, which may not be truly clonal 51 .…”

Section: Discussionmentioning

confidence: 99%

Cell density-dependent differential proliferation of neural stem cells on omnidirectional nanopore-arrayed surface

Kong

Lee

et al. 2017

Sci Rep

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Recently, the importance of surface nanotopography in the determination of stem cell fate and behavior has been revealed. In the current study, we generated polystyrene cell-culture dishes with an omnidirectional nanopore arrayed surface (ONAS) (diameter: 200 nm, depth: 500 nm, center-to-center distance: 500 nm) and investigated the effects of nanotopography on rat neural stem cells (NSCs). NSCs cultured on ONAS proliferated better than those on the flat surface when cell density was low and showed less spontaneous differentiation during proliferation in the presence of mitogens. Interestingly, NSCs cultured on ONAS at clonal density demonstrated a propensity to generate neurospheres, whereas those on the flat surface migrated out, proliferated as individuals, and spread out to attach to the surface. However, the differential patterns of proliferation were cell density-dependent since the distinct phenomena were lost when cell density was increased. ONAS modulated cytoskeletal reorganization and inhibited formation of focal adhesion, which is generally observed in NSCs grown on flat surfaces. ONAS appeared to reinforce NSC-NSC interaction, restricted individual cell migration and prohibited NSC attachment to the nanopore surface. These data demonstrate that ONAS maintains NSCs as undifferentiated while retaining multipotency and is a better topography for culturing low density NSCs.

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“…Treatment with the p160 Rho-associated coiled-coil kinase (ROCK) inhibitor (ROCKi, Y-27632) has been widely used to promote survival of dissociated hESCs after passages [27] and assist EB formation from dissociated single-cell suspension of hESCs [27][28][29]. Nonetheless, ROCKi is a xeno-factor and has been shown to bias cell fate toward residual pluripotency and inhibit differentiation in neural differentiation studies, reducing the utility of the derived cells for potential clinical applications [30]. To facilitate cell aggregation in suspension for EB formation, nonadhesive culture surfaces were developed [12,31], and soluble factors that promote cell-cell interactions were administered in the culture media [32].…”

Section: Conventional Methodsmentioning

confidence: 99%

Engineering Strategies for the Formation of Embryoid Bodies from Human Pluripotent Stem Cells

Pettinato

Wen

Zhang

2015

Stem Cells and Development

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Human pluripotent stem cells (hPSCs) are powerful tools for regenerative therapy and studying human developmental biology, attributing to their ability to differentiate into many functional cell types in the body. The main challenge in realizing hPSC potential is to guide their differentiation in a well-controlled manner. One way to control the cell differentiation process is to recapitulate during in vitro culture the key events in embryogenesis to obtain the three developmental germ layers from which all cell types arise. To achieve this goal, many techniques have been tested to obtain a cellular cluster, an embryoid body (EB), from both mouse and hPSCs. Generation of EBs that are homogeneous in size and shape would allow directed hPSC differentiation into desired cell types in a more synchronous manner and define the roles of cell-cell interaction and spatial organization in lineage specification in a setting similar to in vivo embryonic development. However, previous success in uniform EB formation from mouse PSCs cannot be extrapolated to hPSCs possibly due to the destabilization of adherens junctions on cell surfaces during the dissociation into single cells, making hPSCs extremely vulnerable to cell death. Recently, new advances have emerged to form uniform human embryoid bodies (hEBs) from dissociated single cells of hPSCs. In this review, the existing methods for hEB production from hPSCs and the results on the downstream differentiation of the hEBs are described with emphases on the efficiency, homogeneity, scalability, and reproducibility of the hEB formation process and the yield in terminal differentiation. New trends in hEB production and directed differentiation are discussed.

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Clonal Neural Stem Cells from Human Embryonic Stem Cell Colonies

Cited by 42 publications

References 53 publications

In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells

In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells

Cell density-dependent differential proliferation of neural stem cells on omnidirectional nanopore-arrayed surface

Engineering Strategies for the Formation of Embryoid Bodies from Human Pluripotent Stem Cells

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