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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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Controlling aggregate formation and stability: A. Pre-differentiation improves aggregate formation and stability.hESC cultured on mouse embryonic fibroblast (MEF) feeders were pre-differentiated with 20% serum for 72 hours prior to aggregate formation, resulting an overall reduction in the population level of Oct4 expression [left panel, red line: standard maintenance culture; blue line: pre-differentiated; black: control (unstained)]. Aggregates formed from 2,000 input cells were substantially larger with treatment (blue bar) than without (red bar). Y axis represents aggregate cross-sectional area in microns2, error bars represent one standard deviation. B. The ROCK inhibitor Y-27632 promotes aggregate stability. hESC cells cultured on Matrigel in MEF-conditioned medium with and without pre-differentiation [left panel, red line: standard maintenance culture; blue line: pre-differentiated; black: control (unstained)] were used to form SISO-aggregates in the presence or absence of 10 µM Y-27632. Under these culture conditions, in the absence of both, no aggregates were formed (N.D. - size not determined). With 48 hours pre-differentiation in 20% serum, consistent aggregates were formed (first blue bar). When Y-27632 was added to the suspension of cells without (red bar) or with (second blue bar) pre-differentiation immediately prior to dispensing into the well plate, sizeable aggregates resulted.
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pone-0001565-g002: Controlling aggregate formation and stability: A. Pre-differentiation improves aggregate formation and stability.hESC cultured on mouse embryonic fibroblast (MEF) feeders were pre-differentiated with 20% serum for 72 hours prior to aggregate formation, resulting an overall reduction in the population level of Oct4 expression [left panel, red line: standard maintenance culture; blue line: pre-differentiated; black: control (unstained)]. Aggregates formed from 2,000 input cells were substantially larger with treatment (blue bar) than without (red bar). Y axis represents aggregate cross-sectional area in microns2, error bars represent one standard deviation. B. The ROCK inhibitor Y-27632 promotes aggregate stability. hESC cells cultured on Matrigel in MEF-conditioned medium with and without pre-differentiation [left panel, red line: standard maintenance culture; blue line: pre-differentiated; black: control (unstained)] were used to form SISO-aggregates in the presence or absence of 10 µM Y-27632. Under these culture conditions, in the absence of both, no aggregates were formed (N.D. - size not determined). With 48 hours pre-differentiation in 20% serum, consistent aggregates were formed (first blue bar). When Y-27632 was added to the suspension of cells without (red bar) or with (second blue bar) pre-differentiation immediately prior to dispensing into the well plate, sizeable aggregates resulted.

Mentions: We initially observed significant variability in aggregate formation efficacy between experiments, with nominally replicate trials employing the same cell line and number of cells resulting in coherent aggregate formation after 24 hours in one case, and then failing do so in a subsequent attempt (data not shown). After investigating potential causative factors, we noted that population levels of the pluripotency marker Oct4 seemed to affect aggregate cohesion, with aggregates formed from populations of cells with the highest levels of Oct4 exhibiting the least stability. We thus tested whether differentiation induction using serum-containing medium would improve aggregate stability. As shown in Figure 2A, this approach was successful, with 72 hours of serum induction resulting in a substantial increase in aggregate size and symmetry. hEB yield via this process approaches 100% (one aggregate per well). The introduction of a spin-out step usable in 384-well format without compromising hEB coherence and reproducibility has resulted in a dramatic increase in the numbers of hEB that can be efficiently generated. In combination with a basic liquid dispensing system, we have been able to easily generate several thousand organized hEB in a single experiment; we expect this HTP approach to be limited only by the available liquid-handling technology. The use of steeper well geometries (96- and 384-well V-bottom plates, vs the 96-well U-bottom plates employed in the original report [34]) also contributes to the direct formation of single, symmetrical aggregates in each well.


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)

Controlling aggregate formation and stability: A. Pre-differentiation improves aggregate formation and stability.hESC cultured on mouse embryonic fibroblast (MEF) feeders were pre-differentiated with 20% serum for 72 hours prior to aggregate formation, resulting an overall reduction in the population level of Oct4 expression [left panel, red line: standard maintenance culture; blue line: pre-differentiated; black: control (unstained)]. Aggregates formed from 2,000 input cells were substantially larger with treatment (blue bar) than without (red bar). Y axis represents aggregate cross-sectional area in microns2, error bars represent one standard deviation. B. The ROCK inhibitor Y-27632 promotes aggregate stability. hESC cells cultured on Matrigel in MEF-conditioned medium with and without pre-differentiation [left panel, red line: standard maintenance culture; blue line: pre-differentiated; black: control (unstained)] were used to form SISO-aggregates in the presence or absence of 10 µM Y-27632. Under these culture conditions, in the absence of both, no aggregates were formed (N.D. - size not determined). With 48 hours pre-differentiation in 20% serum, consistent aggregates were formed (first blue bar). When Y-27632 was added to the suspension of cells without (red bar) or with (second blue bar) pre-differentiation immediately prior to dispensing into the well plate, sizeable aggregates resulted.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2215775&req=5

pone-0001565-g002: Controlling aggregate formation and stability: A. Pre-differentiation improves aggregate formation and stability.hESC cultured on mouse embryonic fibroblast (MEF) feeders were pre-differentiated with 20% serum for 72 hours prior to aggregate formation, resulting an overall reduction in the population level of Oct4 expression [left panel, red line: standard maintenance culture; blue line: pre-differentiated; black: control (unstained)]. Aggregates formed from 2,000 input cells were substantially larger with treatment (blue bar) than without (red bar). Y axis represents aggregate cross-sectional area in microns2, error bars represent one standard deviation. B. The ROCK inhibitor Y-27632 promotes aggregate stability. hESC cells cultured on Matrigel in MEF-conditioned medium with and without pre-differentiation [left panel, red line: standard maintenance culture; blue line: pre-differentiated; black: control (unstained)] were used to form SISO-aggregates in the presence or absence of 10 µM Y-27632. Under these culture conditions, in the absence of both, no aggregates were formed (N.D. - size not determined). With 48 hours pre-differentiation in 20% serum, consistent aggregates were formed (first blue bar). When Y-27632 was added to the suspension of cells without (red bar) or with (second blue bar) pre-differentiation immediately prior to dispensing into the well plate, sizeable aggregates resulted.
Mentions: We initially observed significant variability in aggregate formation efficacy between experiments, with nominally replicate trials employing the same cell line and number of cells resulting in coherent aggregate formation after 24 hours in one case, and then failing do so in a subsequent attempt (data not shown). After investigating potential causative factors, we noted that population levels of the pluripotency marker Oct4 seemed to affect aggregate cohesion, with aggregates formed from populations of cells with the highest levels of Oct4 exhibiting the least stability. We thus tested whether differentiation induction using serum-containing medium would improve aggregate stability. As shown in Figure 2A, this approach was successful, with 72 hours of serum induction resulting in a substantial increase in aggregate size and symmetry. hEB yield via this process approaches 100% (one aggregate per well). The introduction of a spin-out step usable in 384-well format without compromising hEB coherence and reproducibility has resulted in a dramatic increase in the numbers of hEB that can be efficiently generated. In combination with a basic liquid dispensing system, we have been able to easily generate several thousand organized hEB in a single experiment; we expect this HTP approach to be limited only by the available liquid-handling technology. The use of steeper well geometries (96- and 384-well V-bottom plates, vs the 96-well U-bottom plates employed in the original report [34]) also contributes to the direct formation of single, symmetrical aggregates in each well.

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