Revolutionizing Medical Research: The Critical Role of Diverse Animal Models with scRNA-seq Technology

Animal testing to advance human health is a complex and often controversial topic, but it remains a crucial aspect of medical research. 

Egyptian, Greek, and Arabic medical texts documented the use of animals for anatomical studies, surgical practice, and drug testing before human trials. This practice continued throughout history, intensifying in the 20th century with the advent of modern science and medicine.

Today, the use of animal models remains crucial for understanding human diseases and developing new treatments. While rodents, particularly mice, have dominated research due to their genetic similarities to humans and ease of manipulation, there’s recognition of a growing need for greater diversity in animal models to improve translational success.

Mice have been the preferred model in medical research since the 1980s.

Their short lifespan, high reproduction rate, and genetic manipulability have made them invaluable. Today, rodent models make up nearly 95% of all laboratory animals. The heavy reliance on mouse models has led to a bias in technological development, data analysis pipelines, and analytical tools, tailored mostly for mice and humans. 

Such reliance on specialized sets of tools limits the adoption of other, more effective animal models. 

In 1972 Dr. Endo and colleagues isolated a promising compound able to inhibit the enzyme that made cholesterol. This first statin worked very well in cultured cells but when tested in rats in 1974 it failed to decrease cholesterol. Only in 1976, when the drug was tested on hens, it reduced the cholesterol by 50% in a month. 

The first statin, if left untested on models other than mice, may have never been discovered.

The estimated time to develop a new drug is 10-15 years, from discovery to market and an overall cost between $450 and $900 million, depending on the drug (Fig. 1). Moreover, 90% of the clinical drugs entering the pipeline fail to reach approval and market. 

The target validation process is challenging due to biological discrepancies among in vitro, animal models, and human disease. Animal models, although useful, do not fully recapitulate human disease. 

The focus on using mouse models has limitations. A 2014 comparative analysis of human and mouse genomes revealed significant differences in gene expression profiles and molecular functions, particularly in immune system-related genes. 

These discrepancies contribute to the high failure rate of drugs in clinical trials, with 40-50% failing due to lack of clinical efficacy. 

Fig.1: A conservative estimation for the overall cost of drug development, starting from preclinical studies and extending to post-market surveillance, suggests that the costs begin at approximately $500 million USD. However, these costs can potentially escalate to over $2 billion USD.

The challenge of effectively translating findings from animal models to human patients is significant. To overcome this challenge, researchers can use alternative animal models that can better mimic human diseases and responses. The growing field of computational biology is making this translation more achievable by enabling the analysis of complex and heterogeneous data.

To generate such data, the technology employed must be functional across a variety of models.

Single-cell RNA sequencing (scRNA-seq) is a cutting edge and versatile technology that can meet this demand. It provides a detailed view of tissue and cellular heterogeneity, enabling the investigation of gene expression dynamics at the single cell level.

There are different scRNA-seq methods, such as droplet-based microfluidics, or plate-based combinatorial barcoding.

Droplet-based methods require hardware investments to function.They can process living cells from any species through a microfluidic system that captures each cell individually and assigns unique identifiers for cell origin tracking. However, the method requires viable cells within a certain size range — to prevent clogging the microfluidics — which reduces the cell types that can be studied, limits the range of species to a narrow subset, and poses challenges for extended research durations.

In contrast, recent advancements have led to an even more versatile, hardware-free technology that works with fixed cells of any animal model, and without the constriction of microfluidic devices. Combinatorial barcoding is truly adaptable across species, cell type, and cell size (Fig 2).

Fig. 2: The Evercode combinatorial barcoding workflow: cells from any sample type are fixed and permeabilized, turning them into their own reaction vessel. The barcoding process then labels cells with an exponentially large number of barcode combinations making it possible to easily scale beyond other technologies.

The following research publications demonstrate the potential discoveries unlocked by employing alternative animal models. 

Scientists employed the Parse Evercode™ assay, a plate-based combinatorial barcoding approach. This technology, not limited by specific models, integrates a protocol for sample fixation, effectively preserving the whole transcriptome. Such integration facilitates the execution of extended or longitudinal studies, while mitigating batch effect concerns. It also allows for deferred sample processing, significantly streamlining the operational demands of long-term studies.

Small Models: Zebrafish and Drosophila Melanogaster

Zebrafish (Danio Rerio) is a valuable model organism for studying human development and disease due to its cost-effectiveness compared to other vertebrate models. Zebrafish are genetically and physiologically similar to humans, sharing 70% of their protein-coding genes. Their transparent embryos develop externally, allowing real-time observation of developmental processes. The zebrafish genome is relatively simple and amenable to genetic editing, making it ideal for investigating multigenic diseases. Zebrafish were the first vertebrate model to demonstrate efficient genome editing using CRISPR in vivo, and they are particularly useful for high-throughput drug screening.

Zebrafish enable real-time monitoring of developmental processes, from embryogenesis to organ formation, within 36 hours post-fertilization. This rapid development facilitates detailed examination of a vaccine’s impact on organ precursors. 

Remarkably, zebrafish have the capacity to regenerate heart cells as its muscle cells can divide, making them an excellent model for cardiomyocyte repair. 

They can also regenerate retinal neurons, restoring visual function, unlike mammals. Understanding the molecular underpinnings of this capability can advance research in human retinal regeneration and vision restoration. A 2022 study in the Journal of Neuroscience examined retinal regeneration in a zebrafish model of inherited retinal degeneration caused by the cep290 gene mutation. Single-cell RNA sequencing (scRNA-seq) revealed sustained expression of Notch3 and other quiescence genes in cep290 mutants, an observation not detected with bulk RNA-seq. This single-cell data was crucial for understanding the molecular basis of failed regeneration in this chronic disease model, highlighting Notch signaling as a key barrier to retinal cell regeneration. This underscores the power of single-cell approaches for dissecting complex tissue responses in disease states.

Similarly, the fruit fly (Drosophila melanogaster), known for its occasional escapes from the fly-room to the neighboring laboratories, has been the cornerstone of genetic research since the beginning of the 20th century. 

Like zebrafish, fruit flies are small, have short reproduction cycles, and are cost-effective to maintain. They share 75% of disease-causing genes with humans. The Notch and Wnt signaling pathways, critical for supporting cellular and tissue development and adult homeostasis, are remarkably conserved between humans and fruit flies, making the latter vital for understanding human disease complexities, including reproductive disorders.

The mushroom body, Drosophila’s brain computational center, is a highly complex neural circuit for associative memory and sensory information processing. The principles of neuronal diversity and complexity in Drosophila have parallels in more complex organisms, including humans. A University of Oregon team used snRNA-seq to explore the diversity of cell types in the brain, focusing on neurons from T2 neuroblasts. They identified over 150 distinct cell clusters, mapped neurotransmitter and neuropeptide expression, and identified unique transcription factor combinations for each cluster. The research mapped the brain atlas with known neuron subtypes to specific clusters, such as olfactory projection, serotonergic, dopaminergic, octopaminergic, mushroom body neurons, supporting the hypothesis that each cluster represents one or a few closely related neuron classes.

This research provides a framework for understanding human brain complexity by elucidating how different neuron types are generated and connected. Understanding the genetic and molecular basis of neuron diversity and connectivity can identify potential therapeutic targets and enhance stem cell-based approaches for brain repair.

Larger Models

While the preference for smaller mammals or non-mammals is due primarily to their ease of use, larger animals have organ systems more similar in size and complexity to those of humans. They more accurately mimic disease progression and provide better information on drug efficacy and safety. 

Chickens 

Chicken embryos are a well-defined developmental model because they develop externally and are large enough to manipulate experimentally. They are accessible for procedures like implantation experiments, and provide insights into conserved developmental mechanisms across vertebrates. 

To understand the effect of the FGF2 molecule on lens development, a team of researchers from Miami University at Oxford, OH, used snRNA-seq on the eye tissue of chicken embryos to profile gene expression in individual lens cells. They utilized a retina regeneration model to assess the effects of the FGF2. They found  a decrease in epithelial cells and changes in intermediate and fiber cell states post FGF2 stimulation. The study also confirms the activation of MAF, a gene influenced by FGF, which is crucial for fiber cell differentiation. Additionally, the study suggests that the retina is a vital source of growth factors, supported by the finding that gene expression levels drop after retinectomy but are restored with FGF2 addition. 

The discovery described in this work could pave the way for novel therapeutic approaches to human retinopathies.

Livestock

Research in livestock reproduction is critical, as it underpins the strength of a critical sector of national economies by bolstering food security and economic strength.

A team from the University of California, Davis, used scRNA-seq to provide insights into the effects of the NANOS3 gene knockout in cattle. NANOS3 is critical in germline development of both sexes, but it is not well characterized. In this long term, longitudinal study, the researchers used cells from 90 day fetal and 283 perinatal gonadal tissues and fixed them before use.

Compared to wild-type, the NANOS3 KO cattle had no germ cells at 283 days, altered gene expression, and possible impairment of somatic gonad development – due to lack of germ cells. These findings demonstrate that NANOS3 is necessary for both male and female fertility in cattle.

Non-Human Primates (NHP)

Non-human primates (NHPs) are essential for translational research because of their close genetic, physiological, and behavioral similarities to humans. They play a crucial role in the development of new therapies, vaccines, and medical devices. NHP models are often essential for preclinical studies to establish the potential efficacy of interventions before they can be tested in human clinical trials.

NHPs are used to study a wide range of  significant human diseases, including HIV/AIDS, tuberculosis, hepatitis, neurodegenerative diseases, diabetes, cardiovascular diseases, respiratory diseases, and vision impairments.

For example, in a longitudinal study, scientists at the Tulane University School of Medicine analyzed the immune response of SIV-infected macaques during a SARS-CoV-2 coinfection. The team found that despite significant immunodeficiency, the SIV-infected macaques did not develop more severe COVID-19 symptoms than their non-SIV-infected counterparts. This challenges the narrative that HIV infection necessarily leads to worse COVID-19 outcomes, suggesting that other factors may be at play in the increased risk observed in some human studies.

The study also revealed aspects of the immune response in coinfected animals. The development of SARS-CoV-2-specific T cell and antibody responses was significantly impaired, a finding that aligns with observations in some humans. This impairment raises critical questions about vaccine efficacy and the risk of reinfection in immunocompromised individuals.

The researchers used scRNA-seq on bronchoalveolar lavage (BAL) cells collected before SARS-CoV-2 inoculation and on days 2, 7, 21, and 28 post-challenge. The researchers fixed the cells and stored them until all samples were collected. The possibility to fix the cells and their whole transcriptome enabled them to perform a comprehensive longitudinal analysis of the immune response in the lungs over the course of SARS-CoV-2 infection. This gave them a granular view of the immune response, highlighting the crucial role of innate immunity, particularly monocytes and macrophages, in controlling SARS-CoV-2 infection when adaptive immunity is compromised. This insight points to new therapeutic approaches for managing COVID-19 in immunocompromised individuals.

Animal research has been a fundamental part of scientific progress throughout history and remains crucial for medical research and drug development.

While mice have been the dominant model for decades, there’s growing recognition of the need for diverse animal models to improve translational success in medical research, including technologies that can easily accommodate other animal models as well.

The advent of scRNA-seq has catalyzed a paradigm shift, enabling unprecedented scrutiny of cellular diversity. This technology’s evolution, particularly through plate-based combinatorial barcoding methods, has significantly enhanced its adaptability. It can now accommodate fixed cells from a multitude of species, thereby supporting extensive and cross-species research endeavors.

While each animal model has its strengths and limitations, greater diversity of species and advanced technologies like scRNA-seq is pushing the boundaries of medical research to enhance the translatability of findings to human health and drug development.

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Explore the publications and datasets that scientists worldwide have published using Parse Biosciences Evercode assays, covering a range of models beyond just mice.