Organoids models to study human vs chimpanzee brain development

Ever since the first report of their generation by Madeline Lancaster (Lancaster et al., Nature 501:373–379; 2013), human brain organoids have been an invaluable tool for developmental neuroscientists. These 3D structures, generated starting from human induced-Pluripotent Stem Cells (hiPSCs), are an ex vivo model of human brain development which can be used to study neurodevelopmental disorders, e.g. microcephaly, amongst other developmental aspects. Noteworthy, organoids give researchers the chance to get insights in the human developing nervous system, otherwise impossible due to ethical implications. After the successful formation of human organoids, scientists obtained also brain organoids from other primates, such as chimpanzee and rhesus macaque.

In a Letter to Nature (Nature 574:418–422; 2019), Kanton and colleagues describe how they generated brain organoids from human, chimpanzee and macaque iPSCs, and subsequently analysed the pool of RNA transcripts (transcriptome) of these organoids at different timepoints, up to 4 months of organoid development. The three main experimental methods employed in this paper are:

• single-cell RNA sequencing (scRNAseq): this technique analyses the RNA transcripts and their variants present in a single cell, in order to determine the regulatory state of gene expression at a cellular level;
• scATACseq: this technique identifies accessible genomic regions in single cells, namely regions with open chromatin in which gene transcription is active;
• single nucleus (sn) RNAseq: this technique analyses the nuclear transcriptome of a single cell.

The data arrays obtained by the researchers at the Max Planck Institute for Evolutionary Anthropology in Leipzig (Germany) have been analysed with different bioinformatic tools. One of these algorithms orders cells on a virtual timeline, the so-called pseudotime. Cells are aligned according to their changes in the transcriptome, which allows the graphic visualisation of the sequence of gene expression and of the developmental and differentiation paths of different cell types. These analyses yielded some interesting discoveries in the context of human organoid development.

Human organoids’ neuronal development is slower than in chimpanzee or rhesus macaque organoids

Within the first 15 days of human organoid preparation, the cells are still in a stem cell-state, with characteristics typical of embryoid bodies, neuroectoderm and neuroepithelium. By month 1, cells have transitioned to a Neural Progenitor Cell (NPC)-state, and by month 2 they have differentiated in cells of different brain areas, including excitatory neurons and inhibitory interneurons; by month 4 there are also astrocytes.

Chimpanzee and macaque organoids pseudotime analysis aligns with the human one at early timepoints but diverges from month 1, when primates’ cells express more astroglial markers and neuronal projection-related genes. At 4 months they also express already upper- and lower-layer cortical neuron specification genes, indicating a faster neural development of non-human, primate organoids.

The analysis of changes in gene expression between human and chimps organoids revealed several human-specific genes that are differentially expressed, meaning they are expressed only in human organoids at a specific timepoint. Such genes are involved in radial-glia proliferation, intermediate progenitors’ proliferation and differentiation, and neuronal migration and maturation. This difference in gene expression highlights the presence of developmental transcriptional programmes specific of human organoids.

Human organoids have different chromatin accessibility, which correlates with the differential gene expression and leads to the transcription of different genes

Human organoids revealed to have different regions of open chromatin as compared to chimps organoids; such regions with different chromatin accessibility correlated with the differentially expressed genes observed. Moreover, regions with differentially accessible chromatin are enriched in Single Nucleotide Changes (SNCs) that are specific to human genes and modify the binding of transcription factors in the coding regions of organoid genes. This divergence in transcriptional activation leads to a divergence in the pseudotimes, with a delay of human organoid development.

Developmental differences between humans and other primates persist in adulthood

The analyses of nuclear transcripts from post-mortem prefrontal cortices of humans, chimps, macaques and bonobo showed that the most conserved genes are those expressed in neurons, whereas astroglial genes have the highest proportion of human-specific differentially expressed genes. Comparing human and chimp gene expression in organoids and adult neurons, interneuronal genes and glutamatergic genes don’t show a strong differential expression.

Overall, this comparative analysis of brain organoid development amongst different primates sheds light on the human-specific regulation of brain development and provides information on differences between humans and chimpanzees. Evolution set apart humans and the great apes around 6 million years ago. Archeological, anthropological and genetic studies constantly bring to light novel insights into the history of our species and the first days of hominidae. However, the overview provided by comparative transcriptional studies such as the one of Kanton and colleagues equips us with a better understanding of what makes us different from our cousins in terms of neuronal development.

 

For the full paper, visit: https://www.nature.com/articles/s41586-019-1654-9

Written by Chiara Galante; Edited by Radhika Menon. Featured image: NGC/Design.