{"id":1002,"date":"2023-02-07T14:11:00","date_gmt":"2023-02-07T14:11:00","guid":{"rendered":"https:\/\/preprod.treefrog.fr\/stemcelljungle\/?p=1002"},"modified":"2023-02-14T11:00:08","modified_gmt":"2023-02-14T11:00:08","slug":"how-to-grow-hipscs-in-lumenized-rosette-conformation-in-the-lab","status":"publish","type":"post","link":"https:\/\/preprod.treefrog.fr\/stemcelljungle\/how-to-grow-hipscs-in-lumenized-rosette-conformation-in-the-lab\/","title":{"rendered":"How to grow hiPSCs in lumenized rosette conformation in the lab ?"},"content":{"rendered":"\t\t
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Over the past decade, developmental biology entered a golden era, fueled by novel biomimetic approaches to culture human pluripotent stem cells (hPSCs). In vitro<\/i> recapitulation of the lumenized rosette conformation was described by the group of Magdalena Zernicka-Goetz in 2014 using mice PSCs cultured in micro-wells coated with Matrigel\u00ae. This approach was then successfully applied to human ES and iPS cells in 2016 by the same team.<\/p>

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With the view of generating hPSC rosettes at scale while automating their maintenance, several groups have explored droplet microfluidics, confining hPSCs in agarose, alginate or PEG, while other teams engineered microfluidic circuits to entrap and grow hPSCs in 3D. While successfully generating lumenized rosettes, such microfluidic systems were primarily designed for academic research, and thus feature limitations either in throughput or total loading capacity.\u00a0<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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Core features of biomimetic approaches <\/b><\/p><\/h3>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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Despite the apparent diversity of in vitro<\/i> approaches for the generation of hPSC lumenized rosettes, key parameters are generally preserved:\u00a0<\/p>

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  • Cells are biophysically contained and a critical aggregate size needs to be reached to form a polarized epithelium with a central lumen (~50-300 \u00b5m).<\/li>
  • Matrigel\u00ae remains the standard extracellular matrix, as it mimics the basal membrane niche and supports PSC polarization and lumenogenesis.<\/li>
  • E8 or mTESR feeder-free media are used, minimizing or eliminating the use of poorly characterized biological components additives such as serum, thus avoiding inconsistencies and variations.<\/li>
  • Hypoxic culture conditions (~ 5% O2) are adopted when possible. Hypoxia was indeed reported to reduce mutation rate and promote polarization in hiPSC cultures.<\/li><\/ul>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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    10 steps to obtain lumenized hPSC colonies (Adapted from Shahbazi et al<\/em>. 2016)\n<\/h3>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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