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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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Conventional hESC differentiation protocols result in heterogeneous aggregates with inconsistent organization and structure: A.Conventional differentiating hESC aggregates, formed by scraping colonies of hESC off the culture surface, are predominantly disordered. As hESC colonies differ widely in size and shape, this heterogeneity is passed on to the differentiating aggregate. Consequently the local microenvironment is neither consistent between nor within the aggregates. Scale bar represents 200 microns. Inset: Fusing aggregates exhibit expression of the transcription factor CDX2 (green, counterstain 7AAD, red) at points of contact, but not elsewhere, demonstrating the ability of microenvironmental cues to override macro-environmental conditions. Scale bar represents 200 microns. B–D. Rare hESC-derived aggregates exhibit self-organization. Within heterogeneous populations of scraped hESC-derived aggregates, a rare subpopulation of hEB can be observed. These hEB are characterized by the presence of two distinct domains, visible in phase contrast (B). An inner domain is positive for the pluripotency marker Oct4 (C/D – red), while the outer domain is positive for the endodermal marker FoxA2 (shown in green in panel C). A laminin-containing membrane at least partially defines the interface between these two domains (shown in green in panel D, counterstained with Hoechst in blue). Scale bars represent 100 microns.
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pone-0001565-g001: Conventional hESC differentiation protocols result in heterogeneous aggregates with inconsistent organization and structure: A.Conventional differentiating hESC aggregates, formed by scraping colonies of hESC off the culture surface, are predominantly disordered. As hESC colonies differ widely in size and shape, this heterogeneity is passed on to the differentiating aggregate. Consequently the local microenvironment is neither consistent between nor within the aggregates. Scale bar represents 200 microns. Inset: Fusing aggregates exhibit expression of the transcription factor CDX2 (green, counterstain 7AAD, red) at points of contact, but not elsewhere, demonstrating the ability of microenvironmental cues to override macro-environmental conditions. Scale bar represents 200 microns. B–D. Rare hESC-derived aggregates exhibit self-organization. Within heterogeneous populations of scraped hESC-derived aggregates, a rare subpopulation of hEB can be observed. These hEB are characterized by the presence of two distinct domains, visible in phase contrast (B). An inner domain is positive for the pluripotency marker Oct4 (C/D – red), while the outer domain is positive for the endodermal marker FoxA2 (shown in green in panel C). A laminin-containing membrane at least partially defines the interface between these two domains (shown in green in panel D, counterstained with Hoechst in blue). Scale bars represent 100 microns.

Mentions: When generated via commonly-employed scraping techniques [11], hEB are extremely heterogeneous in size, shape and organization (Figure 1A). In fact, despite common usage in the hESC field, it is not clear that these objects actually merit the term “embryoid body”, rather than simply “hESC aggregates”. We have thus restricted the use of the term “embryoid body” (EB) to describe aggregates which display clear multi-cellular organization, and use the term “aggregates” or “differentiating aggregates” for the typically-observed hESC-derived structures. This is significant in that no two cells, whether within the same aggregate, or between two aggregates, can be assumed to be experiencing the same microenvironment. As one example, ESC aggregates are prone to adhere to and fuse with one another [25], and we have observed expression of the transcription factor Cdx2 – known to play a significant role in cell fate decisions [26] – in a subset of cells at the junction of fusing hESC aggregates (Figure 1A inset: green – Cdx2, red – DNA), demonstrating that incidental differences in micro-environment can override common macro-environmental signals. Micro-environmental heterogeneity might also be expected to interfere with any intrinsic self-organizational capacity of differentiating hESC. Human ESC-derived aggregate heterogeneity thus places significant limitations on their utility as tools to understand development or control hESC differentiation.


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)

Conventional hESC differentiation protocols result in heterogeneous aggregates with inconsistent organization and structure: A.Conventional differentiating hESC aggregates, formed by scraping colonies of hESC off the culture surface, are predominantly disordered. As hESC colonies differ widely in size and shape, this heterogeneity is passed on to the differentiating aggregate. Consequently the local microenvironment is neither consistent between nor within the aggregates. Scale bar represents 200 microns. Inset: Fusing aggregates exhibit expression of the transcription factor CDX2 (green, counterstain 7AAD, red) at points of contact, but not elsewhere, demonstrating the ability of microenvironmental cues to override macro-environmental conditions. Scale bar represents 200 microns. B–D. Rare hESC-derived aggregates exhibit self-organization. Within heterogeneous populations of scraped hESC-derived aggregates, a rare subpopulation of hEB can be observed. These hEB are characterized by the presence of two distinct domains, visible in phase contrast (B). An inner domain is positive for the pluripotency marker Oct4 (C/D – red), while the outer domain is positive for the endodermal marker FoxA2 (shown in green in panel C). A laminin-containing membrane at least partially defines the interface between these two domains (shown in green in panel D, counterstained with Hoechst in blue). Scale bars represent 100 microns.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2215775&req=5

pone-0001565-g001: Conventional hESC differentiation protocols result in heterogeneous aggregates with inconsistent organization and structure: A.Conventional differentiating hESC aggregates, formed by scraping colonies of hESC off the culture surface, are predominantly disordered. As hESC colonies differ widely in size and shape, this heterogeneity is passed on to the differentiating aggregate. Consequently the local microenvironment is neither consistent between nor within the aggregates. Scale bar represents 200 microns. Inset: Fusing aggregates exhibit expression of the transcription factor CDX2 (green, counterstain 7AAD, red) at points of contact, but not elsewhere, demonstrating the ability of microenvironmental cues to override macro-environmental conditions. Scale bar represents 200 microns. B–D. Rare hESC-derived aggregates exhibit self-organization. Within heterogeneous populations of scraped hESC-derived aggregates, a rare subpopulation of hEB can be observed. These hEB are characterized by the presence of two distinct domains, visible in phase contrast (B). An inner domain is positive for the pluripotency marker Oct4 (C/D – red), while the outer domain is positive for the endodermal marker FoxA2 (shown in green in panel C). A laminin-containing membrane at least partially defines the interface between these two domains (shown in green in panel D, counterstained with Hoechst in blue). Scale bars represent 100 microns.
Mentions: When generated via commonly-employed scraping techniques [11], hEB are extremely heterogeneous in size, shape and organization (Figure 1A). In fact, despite common usage in the hESC field, it is not clear that these objects actually merit the term “embryoid body”, rather than simply “hESC aggregates”. We have thus restricted the use of the term “embryoid body” (EB) to describe aggregates which display clear multi-cellular organization, and use the term “aggregates” or “differentiating aggregates” for the typically-observed hESC-derived structures. This is significant in that no two cells, whether within the same aggregate, or between two aggregates, can be assumed to be experiencing the same microenvironment. As one example, ESC aggregates are prone to adhere to and fuse with one another [25], and we have observed expression of the transcription factor Cdx2 – known to play a significant role in cell fate decisions [26] – in a subset of cells at the junction of fusing hESC aggregates (Figure 1A inset: green – Cdx2, red – DNA), demonstrating that incidental differences in micro-environment can override common macro-environmental signals. Micro-environmental heterogeneity might also be expected to interfere with any intrinsic self-organizational capacity of differentiating hESC. Human ESC-derived aggregate heterogeneity thus places significant limitations on their utility as tools to understand development or control hESC differentiation.

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