RB controls growth, survival, and neuronal migration in human cerebral organoids

Abstract

The tumor suppressor retinoblastoma protein (RB) regulates S-phase cell cycle entry via E2F transcription factors. Knockout (KO) mice have shown that RB plays roles in cell migration, differentiation and apoptosis, in developing and adult brain. In addition, the RB family is required for self-renewal and survival of human embryonic stem cells (hESCs). Since little is known about the role of RB in human brain development, we investigated its function in cerebral organoids differentiated from gene-edited hESCs lacking RB. We show that RB is abundantly expressed in neural stem and progenitor cells in organoids at 15 and 28 days of culture. RB loss promoted S-phase entry in DCX + cells and increased apoptosis in Sox2 + neural stem and progenitor cells, and in DCX + and Tuj1 + neurons. Associated with these cell cycle and pro-apoptotic effects, we observed increased CCNA2 and BAX gene expression, respectively. Moreover, we observed aberrant Tuj1 + neuronal migration in RB-KO organoids and upregulation of the gene encoding VLDLR, a receptor important in reelin signaling. Corroborating the results in RB-KO organoids in vitro, we observed ectopically localized Tuj1 + cells in RB-KO teratomas grown in vivo. Taken together, these results identify crucial functions for RB in the cerebral organoid model of human brain development.

Opportunities for organoids as new models of aging

“…Methods for micron-scale organization of cells within a tissue or microfluidic chamber (Nelson et al, 2008;Todhunter et al, 2015;Kolesky et al, 2016) could be used to create reproducible, high-throughput organoid assays for aging and other diseases. Xiang et al, 2000;van Vliet et al, 2007;Hansen et al, 2010;Eiraku et al, 2011;Qian et al, 2016;Matsui et al, 2017 Gastrointestinal: esophagus, stomach, small intestine, colon Human, mouse Human, mouse, hPSC, mESC Ootani et al, 2009;Barker et al, 2010;Bartfeld et al, 2015;Noguchi et al, 2015;Li et al, 2016 Pancreas Human, mouse Human, mouse, hiPSC Huch et al, 2013;Marciniak et al, 2014;Broutier et al, 2016;Hohwieler et al, 2017 Liver Squires et al, 2003;Caron et al, 2012;Lozito et al, 2013;Bhumiratana et al, 2014 Artery Rat Human, mouse Belmin et al, 1995;L'Heureux et al, 1998 Mammary gland Human, mouse Human Human Villadsen et al, 2007;Linnemann et al, 2011;Tanos et al, 2013;Nguyen-Ngoc et al, 2015 Prostate Human Human, rat Human, mouse Nevalainen et al, 1993;Lang et al, 2001;Karthaus et al, 2014 Ovary, endometrium Human Human Kwong et al, 2009;Turco et al, 2017 Thyroid Human mESC Toda et al, 2002;Antonica et al, 2012 Organoid culture has now been extended to a variety of tissues, including many from adult human primary tissue or cells. h, human; m, mouse; MSC, mesenchymal stem cell.…”

Organ‐on‐chip models: Implications in drug discovery and clinical applications

“…The brain organoid is especially useful in this scenario, as this technology allows researchers to study the human brain as it is developing. One study used CRISPR-Cas9 genome editing to knockout the tumor suppressor retinoblastoma protein (RB) and saw how this increased the size of a developing brain using organoids (Matsui, Nieto-Estevez, Kyrychenko, Schneider, & Hsieh, 2017). Another study cocultured independently patterned organoids to study neuronal migration in a fused cerebral organoid (Bagley, Reumann, Bian, Levi-Strauss, & Knoblich, 2017).…”

Genetic Modification of Brain Organoids

“…It is, however, important to note that NHEJ has also been shown to play an important role in the insertion of transgenes particularly in postmitotic cells which show significantly reduced HDR (Suzuki et al, 2016;Suzuki and Belmonte, 2018). CRISPR/Cas9-mediated genetic modifications are usually conducted on brain organoid founder cells, that is, ESCs and iPSCs (Bershteyn et al, 2017;Iefremova et al, 2017;Li et al, 2017;Matsui et al, 2017;Fiddes et al, 2018;Karzbrun et al, 2018). Cells containing the desired genomic change are then selected and can be used to grow brain organoids.…”

“…One prime application of the CRISPR/Cas9 system in brain organoids is its use in modeling neurodevelopmental and neurodegenerative diseases. Here, either patient mutations can be introduced into control iPS cell lines, or patient iPS cells can be ''repaired'' to generate isogenic controls (Iefremova et al, 2017;Matsui et al, 2017;Fiddes et al, 2018). This was for example successfully applied in the modeling of retinoblastoma (Matsui et al, 2017) and Miller-Dieker Syndrome (Bershteyn et al, 2017;Iefremova et al, 2017).…”

“…In summary, different modes and methods of genetic modifications have been successfully applied at various time points of brain organoid development. The spectrum of potential applications has so far ranged from the simple expression of fluorescent marker proteins (Pasca et al, 2019;Renner et al, 2017) to the study of gene function (Lancaster et al, 2013) to the modeling of disease conditions (Bershteyn et al, 2017;Iefremova et al, 2017;Matsui et al, 2017). Yet, significant untapped potential still remains.…”