Limits...
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.

Show MeSH

Related in: MedlinePlus

SISO-EB are able to self-organize into highly ordered structures.A. Aggregates formed from 2,000 hESC, imaged immediately after recovery (“day 0”)(Ai), or after 1, 2, 3 or 4 days respectively (Aii-Av). Note self organization into an ordered domain (white arrow) and a disordered domain (black arrow), and progressive encirclement of the ordered domain by the disordered domain over time (red arrows). Scale bar represents 500 microns. B. An aggregate fixed on day 3 of differentiation exhibits Oct4 positive nuclei (red) located basally within the cell over a laminin-containing basement membrane (green) in the ordered domain, with morphologically distinct Oct4 negative cells located outside the membrane in the disordered domain (counterstained with Hoechst, blue). Scale bar represents 50 microns. C. A day 5 aggregate exhibits staining for the endodermal marker GATA-6 (green) in the encircling disordered domain, and the pluripotency marker Oct4 (red) within the ordered domain. Note the tendancy of the Oct4 positive nuclei to align along the interface between the two tissue types, and a tendancy towards columnar morphology (white arrow), both characteristics of epiblast tissue, and the absence of mixing between the two cell types. The actin cytoskeleton, probed with phalloidin, is shown in blue. Scale bar represents 50 microns. D–F. Day 3 aggregates, showing staining for the endodermal markers GATA4, GATA6, AFP and FoxA2, the primitive-endoderm marker Sox7, and FGF5, a marker characteristic of epiblast and overlying VE. Scale bars represent 50 microns.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2215775&req=5

pone-0001565-g005: SISO-EB are able to self-organize into highly ordered structures.A. Aggregates formed from 2,000 hESC, imaged immediately after recovery (“day 0”)(Ai), or after 1, 2, 3 or 4 days respectively (Aii-Av). Note self organization into an ordered domain (white arrow) and a disordered domain (black arrow), and progressive encirclement of the ordered domain by the disordered domain over time (red arrows). Scale bar represents 500 microns. B. An aggregate fixed on day 3 of differentiation exhibits Oct4 positive nuclei (red) located basally within the cell over a laminin-containing basement membrane (green) in the ordered domain, with morphologically distinct Oct4 negative cells located outside the membrane in the disordered domain (counterstained with Hoechst, blue). Scale bar represents 50 microns. C. A day 5 aggregate exhibits staining for the endodermal marker GATA-6 (green) in the encircling disordered domain, and the pluripotency marker Oct4 (red) within the ordered domain. Note the tendancy of the Oct4 positive nuclei to align along the interface between the two tissue types, and a tendancy towards columnar morphology (white arrow), both characteristics of epiblast tissue, and the absence of mixing between the two cell types. The actin cytoskeleton, probed with phalloidin, is shown in blue. Scale bar represents 50 microns. D–F. Day 3 aggregates, showing staining for the endodermal markers GATA4, GATA6, AFP and FoxA2, the primitive-endoderm marker Sox7, and FGF5, a marker characteristic of epiblast and overlying VE. Scale bars represent 50 microns.

Mentions: Having established methodologies to generate size-specified hEB at very high frequencies (∼100%), we proceeded to monitor the structure of aggregates for several days following formation, in order to determine the ability of aggregates formed via our protocols to efficiently replicate the self-organization we noted in a rare subpopulation of conventionally-formed aggregate cultures (Figure 1B–D). Aggregates were formed from 2,000 cells, extracted after 24 hours and imaged immediately (day 0) and daily for 4 days. Note the appearance in Figure 5A of two distinct domains in the phase contrast images by day 1 – a highly light-scattering, apparently disordered domain, and a low light-scattering, apparently more ordered domain. The ordered domain exhibits staining for the pluripotency marker Oct4 (Figure 5B and C), while the disordered domain exhibits staining for the endodermal markers GATA-6[48], [49] (Figure 5C and D), GATA4[50] (Figure 5D), AFP[51] and FoxA2[52], [53] (Figure 5E), and the primitive-endoderm marker Sox7[54] (Figure 5F). FGF5, a marker characteristic of epiblast and overlying VE [50], [55], was detected in both (Figure 5F). Indications of epithelial organization are seen in regions where the ordered domain is only a single cell in thickness, with an indication of columnar morphology, and basal localization of nuclei over a laminin-containing basement membrane (Figure 5B and C). These results indicate that SISO-EB self-organize at frequencies approaching unity starting from a hESC culture-derived single-cell suspension. The hEB thus formed recapitulate at least some aspects of early embryogenesis and differentiation, in a manner similar to the previously identified rare EB subpopulation of conventional scraped hESC aggregates (Figure 1B–D) albeit at a much greater efficiency (Figure 5A). Of note, it has recently been reported that organized structures arising from hESC in the context of teratomas are non-clonal in origin – that is, order emerges via the coalescence of multiple cells, rather than being inherited from a single progenitor [10]. In the context of our single-cell-suspension-derived hEB, the rapidity with which these structures form precludes the possibility that they are clonally derived, providing independent confirmation of this observation in an alternative system, and further emphasizing the importance of micro-environment in determining differentiation trajectories.


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-EB are able to self-organize into highly ordered structures.A. Aggregates formed from 2,000 hESC, imaged immediately after recovery (“day 0”)(Ai), or after 1, 2, 3 or 4 days respectively (Aii-Av). Note self organization into an ordered domain (white arrow) and a disordered domain (black arrow), and progressive encirclement of the ordered domain by the disordered domain over time (red arrows). Scale bar represents 500 microns. B. An aggregate fixed on day 3 of differentiation exhibits Oct4 positive nuclei (red) located basally within the cell over a laminin-containing basement membrane (green) in the ordered domain, with morphologically distinct Oct4 negative cells located outside the membrane in the disordered domain (counterstained with Hoechst, blue). Scale bar represents 50 microns. C. A day 5 aggregate exhibits staining for the endodermal marker GATA-6 (green) in the encircling disordered domain, and the pluripotency marker Oct4 (red) within the ordered domain. Note the tendancy of the Oct4 positive nuclei to align along the interface between the two tissue types, and a tendancy towards columnar morphology (white arrow), both characteristics of epiblast tissue, and the absence of mixing between the two cell types. The actin cytoskeleton, probed with phalloidin, is shown in blue. Scale bar represents 50 microns. D–F. Day 3 aggregates, showing staining for the endodermal markers GATA4, GATA6, AFP and FoxA2, the primitive-endoderm marker Sox7, and FGF5, a marker characteristic of epiblast and overlying VE. Scale bars represent 50 microns.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001565-g005: SISO-EB are able to self-organize into highly ordered structures.A. Aggregates formed from 2,000 hESC, imaged immediately after recovery (“day 0”)(Ai), or after 1, 2, 3 or 4 days respectively (Aii-Av). Note self organization into an ordered domain (white arrow) and a disordered domain (black arrow), and progressive encirclement of the ordered domain by the disordered domain over time (red arrows). Scale bar represents 500 microns. B. An aggregate fixed on day 3 of differentiation exhibits Oct4 positive nuclei (red) located basally within the cell over a laminin-containing basement membrane (green) in the ordered domain, with morphologically distinct Oct4 negative cells located outside the membrane in the disordered domain (counterstained with Hoechst, blue). Scale bar represents 50 microns. C. A day 5 aggregate exhibits staining for the endodermal marker GATA-6 (green) in the encircling disordered domain, and the pluripotency marker Oct4 (red) within the ordered domain. Note the tendancy of the Oct4 positive nuclei to align along the interface between the two tissue types, and a tendancy towards columnar morphology (white arrow), both characteristics of epiblast tissue, and the absence of mixing between the two cell types. The actin cytoskeleton, probed with phalloidin, is shown in blue. Scale bar represents 50 microns. D–F. Day 3 aggregates, showing staining for the endodermal markers GATA4, GATA6, AFP and FoxA2, the primitive-endoderm marker Sox7, and FGF5, a marker characteristic of epiblast and overlying VE. Scale bars represent 50 microns.
Mentions: Having established methodologies to generate size-specified hEB at very high frequencies (∼100%), we proceeded to monitor the structure of aggregates for several days following formation, in order to determine the ability of aggregates formed via our protocols to efficiently replicate the self-organization we noted in a rare subpopulation of conventionally-formed aggregate cultures (Figure 1B–D). Aggregates were formed from 2,000 cells, extracted after 24 hours and imaged immediately (day 0) and daily for 4 days. Note the appearance in Figure 5A of two distinct domains in the phase contrast images by day 1 – a highly light-scattering, apparently disordered domain, and a low light-scattering, apparently more ordered domain. The ordered domain exhibits staining for the pluripotency marker Oct4 (Figure 5B and C), while the disordered domain exhibits staining for the endodermal markers GATA-6[48], [49] (Figure 5C and D), GATA4[50] (Figure 5D), AFP[51] and FoxA2[52], [53] (Figure 5E), and the primitive-endoderm marker Sox7[54] (Figure 5F). FGF5, a marker characteristic of epiblast and overlying VE [50], [55], was detected in both (Figure 5F). Indications of epithelial organization are seen in regions where the ordered domain is only a single cell in thickness, with an indication of columnar morphology, and basal localization of nuclei over a laminin-containing basement membrane (Figure 5B and C). These results indicate that SISO-EB self-organize at frequencies approaching unity starting from a hESC culture-derived single-cell suspension. The hEB thus formed recapitulate at least some aspects of early embryogenesis and differentiation, in a manner similar to the previously identified rare EB subpopulation of conventional scraped hESC aggregates (Figure 1B–D) albeit at a much greater efficiency (Figure 5A). Of note, it has recently been reported that organized structures arising from hESC in the context of teratomas are non-clonal in origin – that is, order emerges via the coalescence of multiple cells, rather than being inherited from a single progenitor [10]. In the context of our single-cell-suspension-derived hEB, the rapidity with which these structures form precludes the possibility that they are clonally derived, providing independent confirmation of this observation in an alternative system, and further emphasizing the importance of micro-environment in determining differentiation trajectories.

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