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The effect of vitronectin on the differentiation of embryonic stem cells in a 3D culture system.

ABSTRACT: While stem cell niches in vivo are complex three-dimensional (3D) microenvironments, the relationship between the dimensionality of the niche to its function is unknown. We have created a 3D microenvironment through electrospinning to study the impact of geometry and different extracellular proteins on the development of cardiac progenitor cells (Flk-1(+)) from resident stem cells and their differentiation into functional cardiovascular cells. We have investigated the effect of collagen IV, fibronectin, laminin and vitronectin on the adhesion and proliferation of murine ES cells as well as the effects of these proteins on the number of Flk-1(+) cells cultured in 2D conditions compared to 3D system in a feeder free condition. We found that the number of Flk-1(+) cells was significantly higher in 3D scaffolds coated with laminin or vitronectin compared to colIV-coated scaffolds. Our results show the importance of defined culture systems in vitro for studying the guided differentiation of pluripotent embryonic stem cells in the field of cardiovascular tissue engineering and regenerative medicine.

SUBMITTER: Heydarkhan-Hagvall S

PROVIDER: S-EPMC7731733 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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The effect of vitronectin on the differentiation of embryonic stem cells in a 3D culture system.

Heydarkhan-Hagvall Sepideh S Gluck Jessica M JM Delman Connor C Jung Monica M Ehsani Nazanin N Full Sean S Shemin Richard J RJ

Biomaterials 20111212 7

While stem cell niches in vivo are complex three-dimensional (3D) microenvironments, the relationship between the dimensionality of the niche to its function is unknown. We have created a 3D microenvironment through electrospinning to study the impact of geometry and different extracellular proteins on the development of cardiac progenitor cells (Flk-1(+)) from resident stem cells and their differentiation into functional cardiovascular cells. We have investigated the effect of collagen IV, fibr ...[more]

PMID: 22169822

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Project description:The peripheral hearing process taking place in the cochlea mainly depends on two distinct sensory cell types: the mechanosensitive hair cells and the spiral ganglion neurons (SGNs). The first respond to the mechanical stimulation exerted by sound pressure waves on their hair bundles by releasing neurotransmitters and thereby activating the latter. Loss of these sensorineural cells is associated with permanent hearing loss. Stem cell-based approaches aiming at cell replacement or in vitro drug testing to identify potential ototoxic, otoprotective, or regenerative compounds have lately gained attention as putative therapeutic strategies for hearing loss. Nevertheless, they rely on efficient and reliable protocols for the in vitro generation of cochlear sensory cells for their implementation. To this end, we have developed a differentiation protocol based on organoid culture systems, which mimics the most important steps of in vivo otic development, robustly guiding mouse embryonic stem cells (mESCs) toward otic sensory neurons (OSNs). The stepwise differentiation of mESCs toward ectoderm was initiated using a quick aggregation method in presence of Matrigel in serum-free conditions. Non-neural ectoderm was induced via activation of bone morphogenetic protein (BMP) signaling and concomitant inhibition of transforming growth factor beta (TGF?) signaling to prevent mesendoderm induction. Preplacodal and otic placode ectoderm was further induced by inhibition of BMP signaling and addition of fibroblast growth factor 2 (FGF2). Delamination and differentiation of SGNs was initiated by plating of the organoids on a 2D Matrigel-coated substrate. Supplementation with brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) was used for further maturation until 15 days of in vitro differentiation. A large population of neurons with a clear bipolar morphology and functional excitability was derived from these cultures. Immunostaining and gene expression analysis performed at different time points confirmed the transition trough the otic lineage and final expression of the key OSN markers. Moreover, the stem cell-derived OSNs exhibited functional electrophysiological properties of native SGNs. Our established in vitro model of OSNs development can be used for basic developmental studies, for drug screening or for the exploration of their regenerative potential.

Dataset Information

The effect of vitronectin on the differentiation of embryonic stem cells in a 3D culture system.

Publications

The effect of vitronectin on the differentiation of embryonic stem cells in a 3D culture system.

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