Organoids: Expanding Applications Enabled by Emerging Technologies

The growing need to develop more reliable and human-based model systems are facilitating the rapid development of cell-based culture systems that better mimic the physiological conditions and tissue/organ functions. These systems, derived from primary tissues, embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs) in 3D conditions, are termed as “organoids”.^1,2 The progress of organoid biology is accompanied and driven by the development of biotechnologies and the growing knowledge of cell-niche interactions. For example, the discovery of basement membrane and collagen gels allowed the 3D culture of primary epithelial tissues to form distinct luminal structures.^3,4 Later, with the discovery of ESCs and iPSCs,⁵ their astonishing capability to self-organize were found in 3D aggregation cultures,^6–8 leading to the development of organoid systems representing multiple organ types in humans. More recently, bioengineering tools, such as micropatterning and microfluidics, facilitate more precise 2D and 3D models of early-stage human embryogenesis.^9,10 Improvements in both the complexity of cultured organoids and our understanding towards these organoid systems are continuously progressing with the adoption of new tools such as single-cell transcriptomics, dynamic culture systems, synthetic biomaterials, and flexible electrodes, to mention a few. With the increasing capability to control cellular microenvironment within organoids, the applicability of organoids are expanding as tools to better understand the fundamental developmental processes of organogenesis to disease modeling and drug screening.

Two articles in this special issue focus on the recent advances in bioengineering tools to facilitate the derivation and characterization of organoid systems. Synthetic embryology is an emerging area enabled by the recent advances in bioengineered, artificial hPSC niche. In the review by Chen and Shao,¹¹ the authors presented a thorough review of the development of various stem-cell based models for early-stage embryo development. The authors highlighted the change of paradigm from conventional self-organization based organoid derivation strategy to guided cell organization. Specifically, technologies such as micropatterning, microwells, and microfluidics have been discussed to control geometric boundaries, signaling gradients and tissue-tissue interfaces in organoids. In addition to deriving organoids with higher fidelity, novel technologies also allow for better characterization of organoids. In this issue, Tasnim and Liu presented a timely review on the neural electrophysiology of brain organoids using emergent bioelectronics.¹² A fascinating feature of brain organoids is their capability to generate diverse neurons with electrical activities. It is thus vitally important to record these activities with high spatiotemporal resolution in real-time in a minimally invasive fashion. The authors compared conventional whole-cell patch-clamp recording with new technologies including 2D multi-electrode array (MEA), 3D MEA, and most recently, the 3D flexible bioelectronics, which leads to successful long-term, multiplex recording and detection of network activities. The authors pointed out the need to design new technologies to probe a large number of neurons with cell-type specificity. More intriguing, integration with multimodal sensors and simulators may provide closed-loop feedback to control the development and maturation of brain organoids.

Due to these technological advances improving both the fidelity and cellular diversity in 3D organoids, organoids have been quickly adopted for studies of infectious diseases. Specifically, organoid models have also been applied to study the pathogenic mechanism of SARS-CoV-2, the novel coronavirus that caused a devastating global pandemic. In addition to respiratory syndromes, multi-organ complications contribute significantly to the mortality related to COVID-19. In this timely review, Basu et al. summarized the findings utilizing lung, liver and kidney organoid systems to study the molecular pathogenesis of SARS-CoV-2, which is critical for the development of effective drug targets.¹³ While these systems are powerful tools to investigate and validate virus infection, tropism, immune response gene expression changes, and effects of antiviral drugs, a systemic understanding of SARS-CoV-2 infection requires forming functional connections of various organs. Indeed, organ-on-a-chip or body-on-a-chip technologies are under extensive research to allow researchers to study crosstalk between organs.

Building on this idea of investigating multi-organ crosstalk and cell-type specificity in viral tropism and mechanism of action in organoid systems, Fan et al., presents us with a general framework for setting up studies using brain organoids in infectious diseases.¹⁴ Distinguishing the difference between unguided vs. guided brain organoid differentiation schemes, one has to consider the best fit cellular model for which would be useful for understanding specificity of neurotropism, the ability of viruses to infect certain cell types or brain regions. Using regionalized brain organoids, Fan et al., highlights landmark studies identifying the neurotropism of Zika Virus (ZIKV) and SARS-Cov-2.¹⁴ Interestingly, ZIKV shows specific tropism towards neuroprogenitor cells (radial glia) and astrocytes where SARS-Cov-2 seems to affect choroid plexus specifically. In addition to identifying the mechanism of infection, brain organoids offer a screening tool for testing the safety and efficacy of antiviral drugs as mentioned in the cases of ZIKV and SARS-Cov-2. Still, an unresolved grand challenge remains in teasing apart the infection mechanisms vs. the secondary impact that subsequently affects brain function. Cellular limitations of current brain organoid models still exist including the lack of diverse cell types present in vivo brain such as those cells that arise from mesodermal and endodermal lineages, i.e., vascular cells and blood brain barrier. Incorporation of matrigel that is prominent in brain organoid protocols also presents a challenge as these extracellular matrices interfere with viral infection.

In addition to infectious diseases, Bhattacharya and colleagues¹⁵ highlight the tremendous progress made over the recent years in modeling brain developmental diseases using organoid technology. The reviewed list of a spectrum of neurodevelopmental disorders (NDDs) that has been catalogued and studied using organoids is truly impressive. Overall, studies examining the causality of single gene mutation in the context of brain malformations and early onset childhood NDDs have been most fruitful in interrogating the disease-relevant cellular mechanisms. This wave of success has been attributed to the strength of the current organoid systems to read out clear phenotypes in organoid size, overall structure, proliferation of progenitors, and proportions of cell types within organoids. Even in studies examining idiopathic and clinically heterogeneous NDDs as well as in utero exposure to infectious agents and teratogens have been productive due to the reproducible nature of early cell intrinsic neurodevelopmental programs within these organoids. Importantly, through transcriptomic and epigenetic studies, organoids have taught us the unexpected human-specific cellular features, including the expansion and genetic/epigenetic programs underlying specific human cortical neuroprogenitors (i.e., outer radial glia) that can’t be studied in other non-human models. Emergence of human specific genes, like NOTCH2NL, TMEM14B, TBC1D3, and ARHGAP11B, as well as novel cis-acting enhancers and trans-acting non-coding RNAs highlight the need for better characterization of human specific regulatory signatures that are critical to understanding human specific mechanisms of development and pathogenesis. Lastly, while past studies heavily focused on human cortex and more recently, the interaction between cortex and subcortical regions like basal ganglia and thalamus using assembloids (corticostriatal, -thalamic and -spinal), other brain regional organoids like cerebellar and spinal organoids are emerging, opening exciting avenues for exploring long range migration and connectivity.

Moving forward, in order for organoids to advance to their fullest translational potential as cell-based disease models and modalities in therapeutic design, several aspects should be considered. Most prominent and urgent needs lie in increasing the genetic diversity of iPSC collections and scalability of brain organoids as clinically relevant models. Tegtmeyer and Nehme¹⁶ offer insights into how to consider and improve upon intentionally including ancestry and sex as key wanted biological variables in iPSC research.¹⁷ The authors also offer creative solutions to enhance reproducibility and variability across cell lines and replicates through “cell village” approaches where multiple iPSC lines from diverse donors can be combined for phenotypic discovery and CRISPR based-perturbation screens.^18,19 The utility of disease relevant cell types, regardless of dimensionality, in aiding clinical trials for testing safety and efficacy of drug repurposing and identifying novel drugs are well underway. Cancer biology field has appropriately adopted this model where tumor organoids derived from patient-specific tumors are emerging models to understand tumor heterogeneity and patient-specific drug testing. Sengupta and colleagues reviewed a specific application of patient-derived tumor organoids for studying chromosomal instability (CIN) in gastrointestinal tract cancers.²⁰ CIN is considered as an important factor contributing to the tumor evolution and adaptive resistance to therapies.²¹ While established in 2D culture of cancer cell lines, tumor organoid models can maintain epithelial polarity, recapitulate genetic and epigenetic diversity, and can model immune responses and cancer progression more reliably than 2D models.

From studies mimicking fundamental cellular orchestration of embryonic development to adoption of organoids as high-throughput screening platforms in clinical settings, organoid systems present boundless possibilities. However, limitations in all of these applications are apparent. For example, in the case of current brain organoid protocols, there exists an incomplete representation of cell types derived from mesodermal and endodermal lineages, i.e., microglia, vascular cells and blood brain barrier. Moreover, there is a lack of clear understanding in the cell type identities and subtype specification over maturation period. Potential ways to improve the functional maturation and the survival of diverse cell types could be achieved through improved neural patterning by localized delivery of morphogenic factors and counteracting oxygen/nutrient diffusion limitations via rationally designed engineered microdevices. These advances require creative approaches to address bottlenecks of current technologies. For example, microfluidic culturing systems have limited space and are challenging to provide a dynamic biomechanical environment, and therefore require further modifications for long-term organoid culture. Also, for electrophysiological measurements, it is currently not possible to achieve cell subtype specific measurements for a large number of cells. In addition, the continuous development of new biomaterials as synthetic scaffolds (alternative to Matrigels) with improved biocompatibility and mass transport properties will be needed to improve reproducibility and reduce hypoxia. Together, we envision that the close collaborations between stem cell biologists and bioengineers are greatly needed to push organoid research to the next level.