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 give rise to cardiac, hematopoetic and neural cells.Cardiac differentiation from hEB differentiated for 12 days in suspension culture in serum-containing medium. A) two frames from a video recording of a contractile aggregate, shown before (Ai) and during (Aii) a contraction. Panel Aiii is derived by subtracting panel Ai from panel Aii. The contraction trace (B) was generated by integrating the subtraction image derived from successive frames in the video over the area of contraction, and plotting a 5-point moving average. Neural rosettes (C) were observed after 11 days in culture (4 days in suspension followed by 7 days adherent), staining positive for Pax6 (shown in green) and Sox2 (shown in red). Hematopoetic differentiation was observed after 28 days using Cytospin (D), CFC (E) and flow cytometric (F) assays. G. Quantitative RT-PCR results from aggregates formed from 10,000 pre-differentiated cells in the absence of ROCK inhibition, differentiated for 4 days in suspension followed by an additional 3 days in adherent culture shows down-regulation of pluripotency genes, and up-regulation of markers for endodermal, ectodermal and mesodermal lineages. H. Quantitative RT-PCR results from aggregates formed from 4,000 untreated cells in the presence of ROCK inhibition, differentiated for 4 days in suspension, showed upregulation of mesodermal and endodermal markers, including markers for mesendodermal precursors, but only limited upregulation of the primitive endoderm marker Sox7.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2215775&req=5

pone-0001565-g006: SISO-EB are able to give rise to cardiac, hematopoetic and neural cells.Cardiac differentiation from hEB differentiated for 12 days in suspension culture in serum-containing medium. A) two frames from a video recording of a contractile aggregate, shown before (Ai) and during (Aii) a contraction. Panel Aiii is derived by subtracting panel Ai from panel Aii. The contraction trace (B) was generated by integrating the subtraction image derived from successive frames in the video over the area of contraction, and plotting a 5-point moving average. Neural rosettes (C) were observed after 11 days in culture (4 days in suspension followed by 7 days adherent), staining positive for Pax6 (shown in green) and Sox2 (shown in red). Hematopoetic differentiation was observed after 28 days using Cytospin (D), CFC (E) and flow cytometric (F) assays. G. Quantitative RT-PCR results from aggregates formed from 10,000 pre-differentiated cells in the absence of ROCK inhibition, differentiated for 4 days in suspension followed by an additional 3 days in adherent culture shows down-regulation of pluripotency genes, and up-regulation of markers for endodermal, ectodermal and mesodermal lineages. H. Quantitative RT-PCR results from aggregates formed from 4,000 untreated cells in the presence of ROCK inhibition, differentiated for 4 days in suspension, showed upregulation of mesodermal and endodermal markers, including markers for mesendodermal precursors, but only limited upregulation of the primitive endoderm marker Sox7.

Mentions: To ensure that our new protocols did not interfere with hESC aggregate developmental potential, we next verified that our methodology could be used to generate the specific differentiated lineages typically derived from differentiated hESC aggregates. Differentiated SISO-EB gave rise to beating cardiomyocytes (Figure 6A and B), rosette-forming neural cells (Figure 6C), and CD33+ and CD45+ cells and their associated blood-lineage colony-forming cells (CFC) (Figure 6D–F). Quantitative RT-PCR analysis of hEB formed from 10,000 pre-differentiated cells in the absence of ROCK inhibition, differentiated for 4 days in suspension followed by 3 days adherent culture, showed upregulation of markers of endodermal, ectodermal and mesodermal lineages (Figure 6G) and concomitant reductions (relative to input cells) in the expression of the pluripotency-associated markers Oct4 and Nanog. In a complementary experiment, hEB formed from 4,000 untreated cells in the presence of ROCK inhibition, differentiated for 4 days in suspension, showed upregulation of mesodermal and endodermal markers (Figure 6H). While only a slight increase in levels of Sox7, a marker characteristic of primitive endoderm [54], was observed, much larger increases in other endodermal markers (GATA4, FoxA2 and Sox17) were seen, suggesting the presence of definitive endoderm. This is supported by increased levels of early mesendoderm markers Brachyury and MixL1 indicating the presence of precursor cell types.


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 give rise to cardiac, hematopoetic and neural cells.Cardiac differentiation from hEB differentiated for 12 days in suspension culture in serum-containing medium. A) two frames from a video recording of a contractile aggregate, shown before (Ai) and during (Aii) a contraction. Panel Aiii is derived by subtracting panel Ai from panel Aii. The contraction trace (B) was generated by integrating the subtraction image derived from successive frames in the video over the area of contraction, and plotting a 5-point moving average. Neural rosettes (C) were observed after 11 days in culture (4 days in suspension followed by 7 days adherent), staining positive for Pax6 (shown in green) and Sox2 (shown in red). Hematopoetic differentiation was observed after 28 days using Cytospin (D), CFC (E) and flow cytometric (F) assays. G. Quantitative RT-PCR results from aggregates formed from 10,000 pre-differentiated cells in the absence of ROCK inhibition, differentiated for 4 days in suspension followed by an additional 3 days in adherent culture shows down-regulation of pluripotency genes, and up-regulation of markers for endodermal, ectodermal and mesodermal lineages. H. Quantitative RT-PCR results from aggregates formed from 4,000 untreated cells in the presence of ROCK inhibition, differentiated for 4 days in suspension, showed upregulation of mesodermal and endodermal markers, including markers for mesendodermal precursors, but only limited upregulation of the primitive endoderm marker Sox7.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001565-g006: SISO-EB are able to give rise to cardiac, hematopoetic and neural cells.Cardiac differentiation from hEB differentiated for 12 days in suspension culture in serum-containing medium. A) two frames from a video recording of a contractile aggregate, shown before (Ai) and during (Aii) a contraction. Panel Aiii is derived by subtracting panel Ai from panel Aii. The contraction trace (B) was generated by integrating the subtraction image derived from successive frames in the video over the area of contraction, and plotting a 5-point moving average. Neural rosettes (C) were observed after 11 days in culture (4 days in suspension followed by 7 days adherent), staining positive for Pax6 (shown in green) and Sox2 (shown in red). Hematopoetic differentiation was observed after 28 days using Cytospin (D), CFC (E) and flow cytometric (F) assays. G. Quantitative RT-PCR results from aggregates formed from 10,000 pre-differentiated cells in the absence of ROCK inhibition, differentiated for 4 days in suspension followed by an additional 3 days in adherent culture shows down-regulation of pluripotency genes, and up-regulation of markers for endodermal, ectodermal and mesodermal lineages. H. Quantitative RT-PCR results from aggregates formed from 4,000 untreated cells in the presence of ROCK inhibition, differentiated for 4 days in suspension, showed upregulation of mesodermal and endodermal markers, including markers for mesendodermal precursors, but only limited upregulation of the primitive endoderm marker Sox7.
Mentions: To ensure that our new protocols did not interfere with hESC aggregate developmental potential, we next verified that our methodology could be used to generate the specific differentiated lineages typically derived from differentiated hESC aggregates. Differentiated SISO-EB gave rise to beating cardiomyocytes (Figure 6A and B), rosette-forming neural cells (Figure 6C), and CD33+ and CD45+ cells and their associated blood-lineage colony-forming cells (CFC) (Figure 6D–F). Quantitative RT-PCR analysis of hEB formed from 10,000 pre-differentiated cells in the absence of ROCK inhibition, differentiated for 4 days in suspension followed by 3 days adherent culture, showed upregulation of markers of endodermal, ectodermal and mesodermal lineages (Figure 6G) and concomitant reductions (relative to input cells) in the expression of the pluripotency-associated markers Oct4 and Nanog. In a complementary experiment, hEB formed from 4,000 untreated cells in the presence of ROCK inhibition, differentiated for 4 days in suspension, showed upregulation of mesodermal and endodermal markers (Figure 6H). While only a slight increase in levels of Sox7, a marker characteristic of primitive endoderm [54], was observed, much larger increases in other endodermal markers (GATA4, FoxA2 and Sox17) were seen, suggesting the presence of definitive endoderm. This is supported by increased levels of early mesendoderm markers Brachyury and MixL1 indicating the presence of precursor cell types.

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