Juvenile neuronal ceroid lipofuscinoses (JNCL, also known as Batten disease) is a lysosomal storage disorder associated with fatal neurodegeneration. There is a need for more physiologically relevant human cell-based models to better understand the cellular changes during the disease process and as platforms for drug screening. We used CRISPR/Cas9 and H9 human embryonic stem (ES) cells to reproduce the most common cause of JNCL, a homozygous 1-kb deletion encompassing exons 7 and 8 of the CLN3 gene (CLN3Δex7/8/Δex7/8), with unmodified H9 ES cell line as an isogenic control cell line. We characterised the isogenic CLN3Δex7/8/Δex7/8 cell lines, as well as neurons and cerebral organoids derived from them. Confocal microscopy analysis of the CLN3Δex7/8/Δex7/8 ES cell line showed increased LAMP1 immunoreactivity relative to control cultures, indicating lysosomal enlargement and suggesting waste accumulation. Electron microscopy analysis of CLN3Δex7/8/Δex7/8 organoids at 5-months showed electron dense inclusions characteristics of lipofuscin and increased number of altered mitochondria, compared to controls. Immunohistochemistry of these organoids also revealed higher expression of astrocytes with longer and thicker processes, indicative of reactive astrocytes. These data provide insights on the development of JNCL phenotypes at the pre-symptomatic stage, and identified cellular phenotypes that may be suitable for drug screening.