Micropatterning of human embryonic stem cells dissects the mesoderm and endoderm lineages

Lee, Lawrence Haoran; Peerani, Raheem; Ungrin, Mark; Joshi, Chirag; Kumacheva, Eugenia; Zandstra, PeterW.

doi:10.1016/j.scr.2008.11.004

Cited by 96 publications

(76 citation statements)

References 29 publications

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“…However, simulations limited to bulk concentration effects cannot capture differences in local concentration created by underlying cellular activities. For example, the level of endogenous signaling in ESCs is influenced by local cell density, as shown by controlling cell colony size using micropatterning (4,17,18). We have advanced these previous studies by creating a model and cell culture system to demonstrate that a small set of probabilistic rules for individual ligands can give rise to colony-level cellular responses.…”

Section: Discussionmentioning

confidence: 96%

Predictive microfluidic control of regulatory ligand trajectories in individual pluripotent cells

Moledina

Clarke

Oskooei

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Local (cell-level) signaling environments, regulated by autocrine and paracrine signaling, and modulated by cell organization, are hypothesized to be fundamental stem cell fate control mechanisms used during development. It has, however, been challenging to demonstrate the impact of cell-level organization on stem cell fate control and to relate stem cell fate outcomes to autocrine and paracrine signaling. We address this fundamental problem using a combined in silico and experimental approach in which we directly manipulate, using laminar fluid flow, the local impact of endogenously secreted gp130-activating ligands and their activation of signal transducer and activator of transcription3 (STAT3) signaling in mouse embryonic stem cells (mESC). Our model analysis predicted that flow-dependent changes in autocrine and paracrine ligand binding would impact heterogeneity in cell-and colony-level STAT3 signaling activation and cause a gradient of cell fate determination along the direction of flow. Interestingly, analysis also predicted that local cell density would be inversely proportional to the degree to which endogenous secretion contributed to cell fate determination. Experimental validation using functional activation of STAT3 by secreted factors under microfluidic perfusion culture demonstrated that STAT3 activation and consequently mESC fate were manipulable by flow rate, position in the flow field, and local cell organization. As a unique demonstration of how quantitative control of autocrine and paracrine signaling can be integrated with spatial organization to elicit higher order cell fate effects, this work provides a general template to investigate organizing principles due to secreted factors.Brownian dynamics simulation | embryonic stem cells | leukemia inhibitory factor | cell fate control | cell heterogeneity T he developing embryo uses many spatially and temporally regulated mechanisms of signal propagation, including autocrine, paracrine, and extracellular matrix-mediated signals to control the proliferation and differentiation of progenitor cells. Based on our understanding of in vivo development, in vitro strategies attempt to mimic developmental mechanisms and establish artificial environments or niches that promote specific cell fates. Despite considerable progress in our ability to control pluripotent cell fate in vitro, significant variability and heterogeneity in differentiation protocols exist. One reason for this is thought to be our inability to mimic the temporally and spatially diverse signaling environments that occur in the embryo. Herein we use mouse embryonic stem cells (mESC) cultures as a model system to reveal a role for autocrine and paracrine signaling in cell fate control and to demonstrate how endogenous signaling and cell organization interact to create higher order local cellular environments, or niches.Mouse ESC cultures normally include the soluble interleukin-6 (IL-6) ligand family member leukemia inhibitory factor (LIF). IL-6 ligands such as LIF signal through the Jan...

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Section: Discussionmentioning

confidence: 96%

Predictive microfluidic control of regulatory ligand trajectories in individual pluripotent cells

Moledina

Clarke

Oskooei

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…It has also been shown that manipulating colony size by micropatterning can direct the cells into mesoderm versus endoderm upon differentiation (Lee et al, 2009), which is likely to be a consequence of edge sensing and a reaction-diffusion mechanism. Micropatterning has also been used to study developmental processes beyond gastrulation, namely during the formation and subsequent characterization of cardiomyocytes .…”

Section: Generating Micropatterns: the Role Of Confining External Sigmentioning

confidence: 99%

Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis

2017

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Cells have an intrinsic ability to self-assemble and self-organize into complex and functional tissues and organs. By taking advantage of this ability, embryoids, organoids and gastruloids have recently been generated in vitro, providing a unique opportunity to explore complex embryological events in a detailed and highly quantitative manner. Here, we examine how such approaches are being used to answer fundamental questions in embryology, such as how cells selforganize and assemble, how the embryo breaks symmetry, and what controls timing and size in development. We also highlight how further improvements to these exciting technologies, based on the development of quantitative platforms to precisely follow and measure subcellular and molecular events, are paving the way for a more complete understanding of the complex events that help build the human embryo.

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“…In one approach micro-contact printing is used to pattern ECM onto tissue culture substrates, thereby specifying hESC colony size and geometry (Peerani, Rao et al 2007; Lee, Peerani et al 2009). …”

Section: Pluripotent Stem Cells Hesc Maintenancementioning

confidence: 99%

Geometric Control of Cardiomyogenic Induction in Human Pluripotent Stem Cells

Bauwens

Song

Thavandiran

et al. 2011

Tissue Engineering Part A

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Pluripotent stem cells provide the opportunity to study human cardiogenesis in vitro, and are a renewable source of tissue for drug testing and disease models, including replacement cardiomyocytes that may be a useful treatment for heart failure. Typically, differentiation is initiated by forming spherical cell aggregates wherein an extraembryonic endoderm (ExE) layer develops on the surface. Given that interactions between endoderm and mesoderm influence embryonic cardiogenesis, we examined the impact of human embryonic stem cell (hESC) aggregate size on endoderm and cardiac development. We first demonstrated aggregate size control by micropatterning hESC colonies at defined diameters and transferring the colonies to suspension. The ratio of endoderm (GATA-6) to neural (PAX6) gene and protein expression increased with decreasing colony size. Subsequently, maximum mesoderm and cardiac induction occurred in larger aggregates when initiated with endoderm-biased hESCs (high GATA-6:PAX6), and in smaller aggregates when initiated with neural-biased hESCs (low GATA-6:PAX6). Additionally, incorporating micropatterned aggregates in a stirred suspension bioreactor increased cell yields and contracting aggregate frequency. We next interrogated the relationship between aggregate size and endoderm and cardiac differentiation efficiency in sizecontrolled aggregates, generated using forced aggregation, in defined cardiogenic medium.

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Micropatterning of human embryonic stem cells dissects the mesoderm and endoderm lineages

Cited by 96 publications

References 29 publications

Predictive microfluidic control of regulatory ligand trajectories in individual pluripotent cells

Predictive microfluidic control of regulatory ligand trajectories in individual pluripotent cells

Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis

Geometric Control of Cardiomyogenic Induction in Human Pluripotent Stem Cells

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