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Microfluidic perfusion modulates growth and motor neuron differentiation of stem cell aggregates.

ABSTRACT: Microfluidic technologies provide many advantages for studying differentiation of three-dimensional (3D) stem cell aggregates, including the ability to control the culture microenvironment, isolate individual aggregates for longitudinal tracking, and perform imaging-based assays. However, applying microfluidics to studying mechanisms of stem cell differentiation requires an understanding of how microfluidic culture conditions impact cell phenotypes. Conventional cell culture techniques cannot directly be applied to the microscale, as microscale culture varies from macroscale culture in multiple aspects. Therefore, the objective of this work was to explore key parameters in microfluidic culture of 3D stem cell aggregates and to understand how these parameters influence stem cell behavior and differentiation. These studies were done in the context of differentiation of embryonic stem cells (ESCs) to motor neurons (MNs). We assessed how media exchange frequency modulates the biochemical microenvironment, including availability of exogenous factors (e.g. nutrients, small molecule additives) and cell-secreted molecules, and thereby impacts differentiation. The results of these studies provide guidance on how key characteristics of 3D cell cultures can be considered when designing microfluidic culture parameters. We demonstrate that discontinuous perfusion is effective at supporting stem cell aggregate growth. We find that there is a balance between the frequency of media exchange, which is needed to ensure that cells are not nutrient-limited, and the need to allow accumulation of cell-secreted factors to promote differentiation. Finally, we show how microfluidic device geometries can influence transport of biomolecules and potentially promote asymmetric spatial differentiation. These findings are instructive for future work in designing devices and experiments for culture of cell aggregates.

SUBMITTER: Jackson-Holmes EL

PROVIDER: S-EPMC8102133 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord. To explain our experimental scheme briefly, we are interested in finding target sites for the dimer of transcription factors Isl1 and Lhx3. To mimic the biological activity of Isl1/Lhx3 dimer, we made Isl1-Lhx3 fusion and found that Isl1-Lhx3 has a potent biological activity in multiple systems (i.e. generation of ectopic motor neurons). Then we made ES cell line that induces Flag-tagged Isl1-Lhx3 expression upon Dox treatment. These *mouse* ES cells differentiate to motor neurons (iMN-ESCs) when treated with Dox following EB formation. To identify genomic binding sites of Isl1-Lhx3 (Flag-tagged), we performed ChIP with Flag antibody (pull down of Flag-Isl1-Lhx3) in ES cells treated with Dox. ChIP with Flag antibody in ES cells treated with vehicle (no Dox) was done as a negative control in parallel, and sequenced along with +Dox sample. We have done these experiments twice (two sets).

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Microfluidic perfusion modulates growth and motor neuron differentiation of stem cell aggregates.

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