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ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells.

Pettinato G, Vanden Berg-Foels WS, Zhang N, Wen X - PLoS ONE (2014)

Bottom Line: We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin.Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition.The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America; Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America; Department of Bioengineering, Clemson University, Clemson, South Carolina, United States of America.

ABSTRACT
We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.

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Sizes (the cross-sectional areas, mean ± s.e.m.) over time in suspension culture of hEBs formed under the four different conditions for the (a) BG01V/hOG and (b) H9 cells.
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pone-0100742-g003: Sizes (the cross-sectional areas, mean ± s.e.m.) over time in suspension culture of hEBs formed under the four different conditions for the (a) BG01V/hOG and (b) H9 cells.

Mentions: Next, we evaluated whether ROCKi and/or centrifugation treatment during hEB formation would result in differences in hEB sizes. The sizes (cross-sectional areas) of the hEBs formed under different treatment conditions were measured over time at 2, 4, and 6 days in suspension culture for the two hESC cell lines. For the BG01V/hOG cells, treatment with ROCKi during hEB formation was associated with greater hEB cross-sectional area than those –ROCKi groups when controlling for centrifugation and time (p<0.001) (Fig. 3a). Treatment with centrifugation was also associated with greater hEB cross-sectional area than those no-centrifugation (–spin) groups when controlling for ROCKi and time (p = 0.011). There was a significant synergistic effect of ROCKi and centrifugation treatment on increasing the sizes of resultant hEBs. hESCs treated with both ROCKi and centrifugation (+ROCKi/+spin group) had a greater cross-sectional area than hESCs treated with ROCKi (p<0.001) or centrifugation alone (p<0.001) or untreated hESCs (-ROCKi/-spin group, p<0.001) (detailed model results in Table S1). In all the four treatment groups, the hEB cross-sectional area did not vary over time in suspension culture.


ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells.

Pettinato G, Vanden Berg-Foels WS, Zhang N, Wen X - PLoS ONE (2014)

Sizes (the cross-sectional areas, mean ± s.e.m.) over time in suspension culture of hEBs formed under the four different conditions for the (a) BG01V/hOG and (b) H9 cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4217711&req=5

pone-0100742-g003: Sizes (the cross-sectional areas, mean ± s.e.m.) over time in suspension culture of hEBs formed under the four different conditions for the (a) BG01V/hOG and (b) H9 cells.
Mentions: Next, we evaluated whether ROCKi and/or centrifugation treatment during hEB formation would result in differences in hEB sizes. The sizes (cross-sectional areas) of the hEBs formed under different treatment conditions were measured over time at 2, 4, and 6 days in suspension culture for the two hESC cell lines. For the BG01V/hOG cells, treatment with ROCKi during hEB formation was associated with greater hEB cross-sectional area than those –ROCKi groups when controlling for centrifugation and time (p<0.001) (Fig. 3a). Treatment with centrifugation was also associated with greater hEB cross-sectional area than those no-centrifugation (–spin) groups when controlling for ROCKi and time (p = 0.011). There was a significant synergistic effect of ROCKi and centrifugation treatment on increasing the sizes of resultant hEBs. hESCs treated with both ROCKi and centrifugation (+ROCKi/+spin group) had a greater cross-sectional area than hESCs treated with ROCKi (p<0.001) or centrifugation alone (p<0.001) or untreated hESCs (-ROCKi/-spin group, p<0.001) (detailed model results in Table S1). In all the four treatment groups, the hEB cross-sectional area did not vary over time in suspension culture.

Bottom Line: We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin.Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition.The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America; Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America; Department of Bioengineering, Clemson University, Clemson, South Carolina, United States of America.

ABSTRACT
We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.

Show MeSH