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      Generation of a Purified iPSC-Derived Smooth Muscle-like Population for Cell Sheet Engineering

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          Summary

          Induced pluripotent stem cells (iPSCs) provide a potential source for the derivation of smooth muscle cells (SMCs); however, current approaches are limited by the production of heterogeneous cell types and a paucity of tools or markers for tracking and purifying candidate SMCs. Here, we develop murine and human iPSC lines carrying fluorochrome reporters ( Acta2 hrGFP and ACTA2 eGFP, respectively) that identify Acta2 +/ ACTA2 + cells as they emerge in vitro in real time during iPSC-directed differentiation. We find that Acta2 hrGFP+ and ACTA2 eGFP+ cells can be sorted to purity and are enriched in markers characteristic of an immature or synthetic SMC. We characterize the resulting GFP + populations through global transcriptomic profiling and functional studies, including the capacity to form engineered cell sheets. We conclude that these reporter lines allow for generation of sortable, live iPSC-derived Acta2 +/ ACTA2 + cells highly enriched in smooth muscle lineages for basic developmental studies, tissue engineering, or future clinical regenerative applications.

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          Highlights

          • ACTA2 reporter iPSC lines allow isolation of iPSC-derived SMCs

          • iPSC-SMCs have transcriptomic profile reminiscent of immature or synthetic SMCs

          • Generated iPSC-SMC cell sheets with mechanical properties in physiological ranges

          Abstract

          In this article, Wong, Kotton, and colleagues developed murine and human iPSC lines with fluorochrome reporters for ACTA2, allowing for isolation of putative iPSC-derived smooth muscle-like populations (iPSC-SMC) with transcriptomic profiles reminiscent of immature or synthetic SMCs. The iPSC-SMCs were used to generate cell sheets with mechanical properties within physiological ranges to serve as building blocks for multi-layered tissue constructs.

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          Most cited references36

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          Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells.

          The establishment of the primitive streak and its derivative germ layers, mesoderm and endoderm, are prerequisite steps in the formation of many tissues. To model these developmental stages in vitro, an ES cell line was established that expresses CD4 from the foxa2 locus in addition to GFP from the brachyury locus. A GFP-Bry(+) population expressing variable levels of CD4-Foxa2 developed upon differentiation of this ES cell line. Analysis of gene-expression patterns and developmental potential revealed that the CD4-Foxa2(hi)GFP-Bry(+) population displays characteristics of the anterior primitive streak, whereas the CD4-Foxa2(lo)GFP-Bry(+) cells resemble the posterior streak. Using this model, we were able to demonstrate that Wnt and TGF-beta/nodal/activin signaling simultaneously were required for the generation of the CD4-Foxa2(+)GFP-Bry(+) population. Wnt or low levels of activin-induced a posterior primitive streak population, whereas high levels of activin resulted in an anterior streak fate. Finally, sustained activin signaling was found to stimulate endoderm commitment from the CD4-Foxa2(+)GFP-Bry(+) ES cell population. These findings demonstrate that the early developmental events involved in germ-layer induction in the embryo are recapitulated in the ES cell model and uncover insights into the signaling pathways involved in the establishment of mesoderm and endoderm.
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            Generation of human vascular smooth muscle subtypes provides insight into embryological origin-dependent disease susceptibility

            Heterogeneity of embryological origins is a hallmark of vascular smooth muscle cells (SMCs), which may influence vascular disease development. Differentiation of human pluripotent stem cells (hPSCs) into developmental origin-specific SMC subtypes remains elusive. In this study, we have established a chemically defined protocol where hPSCs were initially induced to form neuroectoderm, lateral plate mesoderm or paraxial mesoderm. These intermediate populations were further differentiated towards SMCs (>80% MYH11+ and ACTA2+) which displayed contractile ability in response to vasoconstrictors and invested perivascular regions in vivo. Derived SMC subtypes recapitulated the unique proliferative and secretory responses to cytokines previously documented in studies using aortic SMCs of distinct origins. Importantly, this system predicted increased extracellular matrix degradation by SMCs derived from lateral plate mesoderm, which was confirmed using rat aortic SMCs from corresponding origins. Collectively, this work will have broad applications in modeling origin-dependent disease susceptibility and in bio-engineered vascular grafts for regenerative medicine.
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              Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells.

              The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.
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                Author and article information

                Contributors
                Journal
                Stem Cell Reports
                Stem Cell Reports
                Stem Cell Reports
                Elsevier
                2213-6711
                15 August 2019
                10 September 2019
                15 August 2019
                : 13
                : 3
                : 499-514
                Affiliations
                [1 ]Center for Regenerative Medicine, Boston University and Boston Medical Center, 670 Albany Street, 2 nd Floor, Boston, MA 02118, USA
                [2 ]Department of Biomedical Engineering, Boston University, 44 Cummington Mall, Boston, MA 02215, USA
                [3 ]The Pulmonary Center and Department of Medicine, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118, USA
                Author notes
                []Corresponding author jywong@ 123456bu.edu
                [∗∗ ]Corresponding author dkotton@ 123456bu.edu
                [4]

                Co-senior author

                Article
                S2213-6711(19)30266-8
                10.1016/j.stemcr.2019.07.014
                6739689
                31422908
                0d04a45b-d6b5-4f8b-b57e-d1c5cf156db6
                © 2019 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 15 February 2019
                : 18 July 2019
                : 19 July 2019
                Categories
                Article

                induced pluripotent stem cells,directed differentiation,smooth muscle cells,cell sheets,fluorochrome reporters

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