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Actin and myosin II modulate differentiation of pluripotent stem cells.

ABSTRACT: Use of stem cell-based therapies in tissue engineering and regenerative medicine is hindered by efficient means of directed differentiation. For pluripotent stem cells, an initial critical differentiation event is specification to one of three germ lineages: endoderm, mesoderm, and ectoderm. Differentiation is known to be regulated by numerous extracellular and intracellular factors, but the role of the cytoskeleton during specification, or early differentiation, is still unknown. In these studies, we used agonists and antagonists to modulate actin polymerization and the actin-myosin molecular motor during spontaneous differentiation of embryonic stem cells in embryoid bodies. We found that inhibiting either actin polymerization or actin-myosin interactions led to a decrease in differentiation to the mesodermal lineage and an increase in differentiation to the endodermal lineage. Thus, targeting processes that regulate cytoskeletal tension may be effective in enhancing or inhibiting differentiation towards cells of the endodermal or mesodermal lineages, which include hepatocytes, islets, cardiomyocytes, endothelial cells, and osteocytes. Therefore, these fundamental findings demonstrate that modulation of the cytoskeleton may be useful in production for a range of cell-based therapies, including for liver, pancreatic, cardiac, vascular, and orthopedic applications.

SUBMITTER: Boraas LC

PROVIDER: S-EPMC5903644 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Actin and myosin II modulate differentiation of pluripotent stem cells.

Boraas Liana C LC Pineda Emma T ET Ahsan Tabassum T

PloS one 20180417 4

Use of stem cell-based therapies in tissue engineering and regenerative medicine is hindered by efficient means of directed differentiation. For pluripotent stem cells, an initial critical differentiation event is specification to one of three germ lineages: endoderm, mesoderm, and ectoderm. Differentiation is known to be regulated by numerous extracellular and intracellular factors, but the role of the cytoskeleton during specification, or early differentiation, is still unknown. In these studi ...[more]

PMID: 29664925

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Project description:Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/β-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

Dataset Information

Actin and myosin II modulate differentiation of pluripotent stem cells.

Publications

Actin and myosin II modulate differentiation of pluripotent stem cells.

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