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Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates.

Ungrin MD, Joshi C, Nica A, Bauwens C, Zandstra PW - PLoS ONE (2008)

Bottom Line: Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions.Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis.These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Background: Human embryonic stem cells (hESC) should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However, because the three-dimensional hESC aggregates [embryoid bodies (hEB)] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation, progress in hESC technology development has been hindered.

Methodology/principal findings: Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm(2).

Conclusions/significance: The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP) approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.

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Related in: MedlinePlus

SISO-aggregation allows for the generation of size-controlled aggregates.(A) hEB were generated by scraping, and SISO-aggregates were generated from input populations of 400, 2,000 and 10,000 cells in 384-well plates, and recovered by centrifugation. After imaging in phase-contrast mode, images were thresholded and cross-sectional areas were calculated using the ImageJ software package. Values obtained were extremely consistent, with coefficients of variation of 0.09, 0.06 and 0.08 respectively, vs 0.72 for the scraped hEB. (B) The base-10 logarithm of cross sectional area is plotted on a histogram, demonstrating the clear separation between aggregate sizes and dramatic increase in size control over scraping techniques.
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pone-0001565-g003: SISO-aggregation allows for the generation of size-controlled aggregates.(A) hEB were generated by scraping, and SISO-aggregates were generated from input populations of 400, 2,000 and 10,000 cells in 384-well plates, and recovered by centrifugation. After imaging in phase-contrast mode, images were thresholded and cross-sectional areas were calculated using the ImageJ software package. Values obtained were extremely consistent, with coefficients of variation of 0.09, 0.06 and 0.08 respectively, vs 0.72 for the scraped hEB. (B) The base-10 logarithm of cross sectional area is plotted on a histogram, demonstrating the clear separation between aggregate sizes and dramatic increase in size control over scraping techniques.

Mentions: Having established a robust protocol for hEB formation in a defined medium formulation, we sought to quantify our ability to regulate aggregate size. Aggregates were formed in 384-well plates from 400, 2,000 and 10,000 cells per well, and spun out after 24 h. Cross-sectional area data was collected via phase-contrast microscopy, and quantified using the ImageJ software package. The improvement in aggregate consistency and size control over conventional scraping techniques is striking (Figure 3), with coefficients of variation of less than 0.1 for all three SISO aggregate sizes, compared to >0.7 for the scraped hEB. From previously published work [34] and our own investigations (data not shown), hEB size plays a significant role in establishing differentiation outcomes; thus the ability to control this parameter is important to fundamental and applied applications.


Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates.

Ungrin MD, Joshi C, Nica A, Bauwens C, Zandstra PW - PLoS ONE (2008)

SISO-aggregation allows for the generation of size-controlled aggregates.(A) hEB were generated by scraping, and SISO-aggregates were generated from input populations of 400, 2,000 and 10,000 cells in 384-well plates, and recovered by centrifugation. After imaging in phase-contrast mode, images were thresholded and cross-sectional areas were calculated using the ImageJ software package. Values obtained were extremely consistent, with coefficients of variation of 0.09, 0.06 and 0.08 respectively, vs 0.72 for the scraped hEB. (B) The base-10 logarithm of cross sectional area is plotted on a histogram, demonstrating the clear separation between aggregate sizes and dramatic increase in size control over scraping techniques.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2215775&req=5

pone-0001565-g003: SISO-aggregation allows for the generation of size-controlled aggregates.(A) hEB were generated by scraping, and SISO-aggregates were generated from input populations of 400, 2,000 and 10,000 cells in 384-well plates, and recovered by centrifugation. After imaging in phase-contrast mode, images were thresholded and cross-sectional areas were calculated using the ImageJ software package. Values obtained were extremely consistent, with coefficients of variation of 0.09, 0.06 and 0.08 respectively, vs 0.72 for the scraped hEB. (B) The base-10 logarithm of cross sectional area is plotted on a histogram, demonstrating the clear separation between aggregate sizes and dramatic increase in size control over scraping techniques.
Mentions: Having established a robust protocol for hEB formation in a defined medium formulation, we sought to quantify our ability to regulate aggregate size. Aggregates were formed in 384-well plates from 400, 2,000 and 10,000 cells per well, and spun out after 24 h. Cross-sectional area data was collected via phase-contrast microscopy, and quantified using the ImageJ software package. The improvement in aggregate consistency and size control over conventional scraping techniques is striking (Figure 3), with coefficients of variation of less than 0.1 for all three SISO aggregate sizes, compared to >0.7 for the scraped hEB. From previously published work [34] and our own investigations (data not shown), hEB size plays a significant role in establishing differentiation outcomes; thus the ability to control this parameter is important to fundamental and applied applications.

Bottom Line: Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions.Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis.These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Background: Human embryonic stem cells (hESC) should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However, because the three-dimensional hESC aggregates [embryoid bodies (hEB)] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation, progress in hESC technology development has been hindered.

Methodology/principal findings: Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm(2).

Conclusions/significance: The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP) approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.

Show MeSH
Related in: MedlinePlus