Establishment of human fetal hepatocyte organoids and CRISPR-Cas9-based gene knockin and knockout in organoid cultures from human liver

Delilah Hendriks, Benedetta Artegiani, Huili Hu, Susana Chuva de Sousa Lopes, Hans Clevers

Onderzoeksoutput: Bijdrage aan wetenschappelijk tijdschrift/periodieke uitgaveArtikelWetenschappelijkpeer review

Samenvatting

The liver is composed of two epithelial cell types: hepatocytes and liver ductal cells. Culture conditions for expansion of human liver ductal cells in vitro as organoids were previously described in a protocol; however, primary human hepatocytes remained hard to expand, until recently. In this protocol, we provide full details of how we overcame this limitation, establishing culture conditions that facilitate long-term expansion of human fetal hepatocytes as organoids. In addition, we describe how to generate (multi) gene knockouts using CRISPR-Cas9 in both human fetal hepatocyte and adult liver ductal organoid systems. Using a CRISPR-Cas9 and homology-independent organoid transgenesis (CRISPR-HOT) approach, efficient gene knockin can be achieved in these systems. These gene knockin and knockout approaches, and their multiplexing, should be useful for a variety of applications, such as disease modeling, investigating gene functions and studying processes, such as cellular differentiation and cell division. The protocol to establish human fetal hepatocyte organoid cultures takes ~1-2 months. The protocols to genome engineer human liver ductal organoids and human fetal hepatocyte organoids take 2-3 months.

Originele taal-2Engels
TijdschriftNature Protocols
DOI's
StatusE-pub ahead of print - 27 nov 2020

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