The use of agarose microwells for scalable embryoid body formation and cardiac differentiation of human and murine pluripotent stem cells

Dahlmann, Julia; Kensah, George; Kempf, Henning; Skvorc, David; Gawol, Anke; Elliott, David A.; Dräger, Gerald; Zweigerdt, Robert; Martin, Ulrich; Gruh, Ina

doi:10.1016/j.biomaterials.2012.12.024

Cited by 132 publications

(142 citation statements)

References 31 publications

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“…4,19,20 A number of different methods for EB formation and differentiation exist, with recent improvements in controlled EB formation and maintenance of uniform sizes achieved by microtechnologies alone or in combination with hydrodynamic culture systems. 13,21,22 Regardless of the specific methods used, EBs generally exhibit a similar loss of pluripotent markers before progressive expression of differentiated phenotypes is observed. Accordingly, our results suggest a pattern of ECM and GF gene expression that temporally corresponds well to the known sequence of embryonic developmental stages.…”

Section: Discussionmentioning

confidence: 99%

Differential Expression of Extracellular Matrix and Growth Factors by Embryoid Bodies in Hydrodynamic and Static Cultures

Fridley

Nair

McDevitt

2014

Tissue Engineering Part C: Methods

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During development, cell fate specification and tissue development are orchestrated by the sequential presentation of soluble growth factors (GF) and extracellular matrix (ECM) molecules. Similarly, differentiation of stem cells in vitro relies upon the temporal presence of extracellular cues within the microenvironment. Hydrodynamic culture systems are not limited by volume restrictions and therefore offer several practical advantages for scalability over static cultures; however, hydrodynamic cultures expose cells to physical parameters not present in static culture, such as fluid shear stress and mass transfer through convective forces. In this study, the differences between static and hydrodynamic culture conditions on the expression of ECM and GF molecules during the differentiation of mouse embryonic stem cells were examined at both the gene and protein level. The expression of ECM and GF genes exhibited an early decrease in static cultures based on heat map and hierarchical clustering analysis and a relative delayed increase in hydrodynamic cultures. Although the temporal patterns of specific ECM and GF protein expression were comparable between static and hydrodynamic cultures, several notable differences in the magnitudes of expression were observed at similar time points. These results describe the establishment of an analytical framework that can be used to examine the expression patterns of ECM and GF molecules expressed by pluripotent stem cells undergoing differentiation as 3D multicellular aggregates under different culture conditions, and suggest that physical parameters of stem cell microenvironments can alter endogenous ECM and GF expression profiles that may, in turn, influence cell fate decisions.

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Section: Discussionmentioning

confidence: 99%

Differential Expression of Extracellular Matrix and Growth Factors by Embryoid Bodies in Hydrodynamic and Static Cultures

Fridley

Nair

McDevitt

2014

Tissue Engineering Part C: Methods

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“…For instance, the hESC lines derived between 2001 and today exhibit substantial differences in behavior. Indeed, the spatial-temporal stage, from which the cells from the inner cell mass were extracted, has resulted in a predisposition of differentiation toward a specific germ layer (ectoderm, mesoderm, or endoderm) based upon the theory of [3,12,28,29,37,69,70,73] EB, embryoid body; hPSC, human pluripotent stem cell; bFGF, basic fibroblast growth factor; LIF, leukemia inhibitory factor; HD, hanging drop; hESC, human embryonic stem cell; ROCKi, Rhoassociated coiled-coil kinase inhibitor; hEBs, human embryoid bodies; PDMS, polydimethylsiloxane.…”

Section: Discussionmentioning

confidence: 99%

“…In the microwell approach, microwells/microcavities of a volume in the range of several microliters were created on nonadherent, micropatterned polydimethylsiloxane (PDMS) surfaces to allow controlled cell aggregation and formation of one discrete EB per microwell [69] (Fig. 9).…”

Section: Figmentioning

confidence: 99%

“…Alternatively, low-cost, time-efficient, in-house mass production methods of microwell plates using off-theshelf materials are desirable. Attempts have been directed toward creating nonadhesive agarose/Dulbecco's modified Eagle's medium (DMEM) microwells using soft lithography strategy from different templates [69]. Agarose is desirable for microwell fabrication, attributing to its moldability, transparency, and noncell-adhesive properties.…”

Section: Figmentioning

confidence: 99%

“…Another important factor of cell aggregation is the geometry of the microwells. It was found that an angled geometry without horizontal interspaces was preferable since cells settling on horizontal interspaces may not contribute to aggregation and therefore die from anoikis [69]. Using the same technique, EB formation from murine PSCs was much easier when compared with that of hPSCs [69].…”

Section: Figmentioning

confidence: 99%

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Engineering Strategies for the Formation of Embryoid Bodies from Human Pluripotent Stem Cells

Pettinato

Wen

Zhang

2015

Stem Cells and Development

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Human pluripotent stem cells (hPSCs) are powerful tools for regenerative therapy and studying human developmental biology, attributing to their ability to differentiate into many functional cell types in the body. The main challenge in realizing hPSC potential is to guide their differentiation in a well-controlled manner. One way to control the cell differentiation process is to recapitulate during in vitro culture the key events in embryogenesis to obtain the three developmental germ layers from which all cell types arise. To achieve this goal, many techniques have been tested to obtain a cellular cluster, an embryoid body (EB), from both mouse and hPSCs. Generation of EBs that are homogeneous in size and shape would allow directed hPSC differentiation into desired cell types in a more synchronous manner and define the roles of cell-cell interaction and spatial organization in lineage specification in a setting similar to in vivo embryonic development. However, previous success in uniform EB formation from mouse PSCs cannot be extrapolated to hPSCs possibly due to the destabilization of adherens junctions on cell surfaces during the dissociation into single cells, making hPSCs extremely vulnerable to cell death. Recently, new advances have emerged to form uniform human embryoid bodies (hEBs) from dissociated single cells of hPSCs. In this review, the existing methods for hEB production from hPSCs and the results on the downstream differentiation of the hEBs are described with emphases on the efficiency, homogeneity, scalability, and reproducibility of the hEB formation process and the yield in terminal differentiation. New trends in hEB production and directed differentiation are discussed.

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Modulation of Stem Cells Behavior Through Bioactive Surfaces

Gomes

Assunção‐Silva

Sousa

et al. 2016

Advanced Surfaces for Stem Cell Research

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The use of agarose microwells for scalable embryoid body formation and cardiac differentiation of human and murine pluripotent stem cells

Cited by 132 publications

References 31 publications

Differential Expression of Extracellular Matrix and Growth Factors by Embryoid Bodies in Hydrodynamic and Static Cultures

Differential Expression of Extracellular Matrix and Growth Factors by Embryoid Bodies in Hydrodynamic and Static Cultures

Engineering Strategies for the Formation of Embryoid Bodies from Human Pluripotent Stem Cells

Modulation of Stem Cells Behavior Through Bioactive Surfaces

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