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Organoids and single cell sequencing – opening the doors to personalised medicine

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Case studies Neurosciences Review Stem Cell Research

Organoids and single cell sequencing – opening the doors to personalised medicine

The employment of animal models and single cell cultures in biomedical research has contributed greatly to understanding various diseases, from auto-immune to cancer, as well as drug and vaccine development. The abundant use of these model systems is proof of their value in research laboratories towards ground-breaking discoveries. The process of using animal models to investigate disease mechanisms is initiated by examining biological processes through genetic screens in invertebrates. Subsequently, evolutionary conservation is analysed in mammalian model systems, ultimately leading to clinical translation to humans. This common practice has led to innovative discoveries in identifying the pathological mechanisms of various human diseases. However, even with these advanced methods, the translation of model systems to humans is still a limiting factor in the drug discovery process. Differences in cellular context, physiology and genetics of different species contribute to questioning the legitimacy of these systems and can influence how accurately the human response can be predicted. Although rare, cases of failure to predict human pharmacodynamics and toxicity from pre-clinical animal studies, have unfortunately occurred (1). Therefore, the development of organoids, a human in vitro 3D cell culture system, derived from stem cells, offers an attractive alternative to overcome these limitations.

The recent developments in stem cell biology have allowed the growth of human tissues to resemble organs in vitro. Established from either pluripotent stem cells (PSC) or adult stem cells (ASC) from resected or biopsied organs, these self-organising systems, cultured on an extra cellular matrix, connects the gap between single cell culture and in vivo models. Organoids offer the ability to replicate the complexity of an organ, to recapitulate the function, cellular components, and tissue architecture of the original organ. Key advantages of organoids include their genetic stability over long periods of time (years) and reduced expression of stress response genes during extended culture (2). Additional to the high similarities of human tissues, the same experimental approaches and analytical techniques developed for cell lines, including fluorescent labelling, live cell imaging and mass spectrometry can also be applied to organoids (3). Organoids developed from ASCs are extracted from healthy or tumourous tissue biopsies and processed into single cell suspensions before embedment onto an extracellular matrix. A cocktail of specific growth factors to initiate cell differentiation are applied regularly until the organoids have expanded (Figure 1). Organoids cultured from human ASCs includes intestine, liver, stomach, pancreas, and colon (4-7). PSC derived organoids are grown from 2D cultures of PSCs that mature into spheroids. Following this they are embedded onto an extracellular matrix and matured with growth factors specific to the desired tissue (Figure 1). Cellular architecture is determined by the original tissue, ultimately influencing the configuration of the organoid. Liver, intestine, lung, kidney, and stomach organoids can be cultured from PSCs (8-12).

Figure 1. Schematic overview of PSC and ASC derived organoids and their use in combination with single cell sequencing technology for various applications from modelling diseases to discovering novel marker genes.

The rapid development of single cell sequencing analysis and technology has unlocked the potential to investigate the heterogeneity of cells; to understand molecular mechanisms and developmental processes; and to investigate different disease states at a single cell resolution. The exponential interest to incorporate this technology into current research has advanced single cell methods, enabling high-throughput processing of thousands of cells, and progress to single cell multi-omic analysis. Incorporation of microfluidic devices in combination with bar-coding beads, into single cell technologies, facilitates the capacity to simultaneously obtain data from a significant number of cells. Singleron’s core technology is based upon microfluidic microwell chips that together with bar coding beads captures mRNA from tens of thousands of cells for processing, to provide crucial information on the origin, function, and individual variation for different cell types.

In recent years, studies have been published combining single cell sequencing and organoid technologies to model organ development, tissue regeneration and diseases, emphasising a potential to synchronise these revolutionary techniques. Joining these technologies paves a novel approach to discover rare cell types and gene markers, delineate cell differentiation pathways, model diseases, and replicate heterogeneity of cells in bodily organs (Figure 1).

Various studies have demonstrated, using single cell RNA sequencing (scRNA-seq) and organoids as a powerful tool to discover novel cell types in organs. For example, a study analysing the cellular composition of the intestinal epithelium, sequenced 238 cells from intestinal organoids (14). The authors identified three novel subtypes of enteroendocrine cells, in addition to, Reg4 as a gene marker for enteroendocrine cells (14). Other studies used scRNA-seq and adult lung organoids to reveal Lgr5+ and Lgr6+ as gene markers of mesenchymal cells (15), while another study identified a subtype of progenitor-like acinar cells expressing progenitor markers with the ability for self-renewal from pancreas organoids (16).

The unique single cell information of distinct cell types from an organ obtained from scRNA-seq, is beneficial to reveal computational delineation of differentiation pathways. Utilising human cerebrum and cerebral organoids, together with an algorithm to analyse cell lineage relationships called Monocle, a path connecting apical progenitors to neurons via EOMES-expressing basal progenitors was uncovered (17).

Cellular heterogeneity is key to understanding the pathology and developmental process of a disease, where combining organoids and scRNA-seq have enhanced the ability to investigate this. scRNA-seq of 3D liver bud organoids revealed extensive similarities between them and foetal liver cells, in addition to, the ability for recapitulating hepatic, stromal and endothelial interactions (19). Other publications have reported single cell sequencing of human brain cerebral cortical spheroids cultures containing astrocyte-lineage cells to discover a striking similarity to native brain cells (20).

Disease mechanisms can originate from a distinct subpopulation of cells. Examining specific roles of individual cell populations in disease models, such as organoids, unlocks the potential for precision medicine in treating certain diseases. Studies using scRNA-seq to investigate bacterial infection using Salmonella- and Heligmosomoides polygyrus infected intestinal organoids, identified Salmonella infection to instigate accumulation of secretory cell types, while Heligmosomoides polygyrus infection results in accumulation of absorptive enterocytes and Paneth cells (19). Another publication using cerebral organoids cultured from Miller-Dieker syndrome induced pluripotent stem cells, reported a defect in mitosis in the outer radial glial (20).

Combining scRNA-seq and organoid technologies is advancing research in creating models for development, regeneration, and disease, paving an innovative method in developing personalised medicine. The technological advances in scRNA seq has enriched the quality, efficiency, and accuracy of analysis, encouraging the simultaneous use of scRNA seq and organoids to research the disease and development in organs.

To learn more about how Singleron’s technology can advance your research on organoids, contact us at info@singleronbio.com.

References

  1. Philip Chaikin. The Bial 10-2474 phase 1 study — a drug development perspective and recommendations for future first-in-human trials. J Clin Pharmacol, 2017. DOI: 10.1002/jcph.889
  2. Sato T et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature, 2009. DOI: 10.1038/nature07935
  3. Cristobal A et al. Personalized proteome profiles of healthy and tumor human colon organoids reveal both individual diversity and basic features of colorectal cancer. Cell Rep, 2017. DOI: 10.1016/j.celrep.2016.12.016
  4. Sato T et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology, 2011. DOI: 10.1053/j.gastro.2011.07.050
  5. Huch M et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature, 2013b. DOI: 10.1038/nature11826
  6. Huch M et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J, 2013a. DOI: 10.1038/emboj.2013.204
  7. Barker N et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell, 2010. DOI: 10.1016/j.stem.2009.11.013
  8. Spence JR et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature, 2011. DOI: 10.1038/nature09691
  9. Takebe T et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature, 2013. DOI: 10.1038/nature12271
  10. Dye BR et al. In vitro generation of human pluripotent stem cell derived lung organoids. eLife, 2015. DOI: 10.7554/eLife.05098
  11. Takasato M et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature, 2015. DOI: 10.1038/nature15695
  12. McCracken KW et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature, 2014. DOI: 10.1038/nature13863
  13. Quadrato G et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature, 2017. DOI: 10.1038/nature22047
  14. Grun D et al. Single-cell messenger RNA sequencing reveals rare intestinal cell types. Nature, 2015. DOI: 10.1038/nature14966
  15. Lee JH et al. Anatomically and functionally distinct lung mesenchymal populations marked by Lgr5 and Lgr6. Cell, 2017. DOI: 10.1016/j.cell.2017.07.028
  16. Wollny D et al. Single-cell analysis uncovers clonal acinar cell heterogeneity in the adult pancreas.Dev Cell, 2016. DOI: 10.1016/j.devcel.2016.10.002
  17. Camp JG et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proc Natl Acad Sci, 2015. DOI: 10.1073/pnas.1520760112
  18. Sloan SA et al. Human astrocyte maturation captured in 3D cerebral cortical spheroids derived from pluripotent stem cells. Neuron, 2017. DOI: 10.1016/j.neuron.2017.07.035
  19. Haber AL et al. A single-cell survey of the small intestinal epithelium. Nature, 2017. DOI: 10.1038/nature24489
  20. Bershteyn M et al. Human iPSC-derived cerebral organoids model cellular features of lissencephaly and reveal prolonged mitosis of outer radial glia. Cell Stem Cell, 2017. DOI: 10.1016/j.stem.2016.12.007
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