Bowdoin Science Journal

Vincent Chen '27

Expanding Brain Cells: Improvements in Molecular Expansion Technology a Possible Milestone in Neuropathology Diagnosis

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The immune system identifies cells in the body by examining molecular stages on the surface of cells, which are known as antigens. Both the human body and mind utilize this information to decide if cells within our body pose a threat to our well-being. Nevertheless, science today continues to struggle with differentiating harmless and harmful cells. For example, cancerous cells may be so similar in antigen identities that they can’t be told apart. However, with a deeper look, researchers may be able to uncover smaller antigens that allows humanity to successfully distinguish between such cells.

Scientists have been using technology that expands cells in uniform across three dimensions to identify such smaller antigens that have previously been unaccounted for. The most recent advancement involved dExPath (decrowding expansion pathology), an improvement with four times greater expansions and stronger resolutions compared to its predecessor, ExPath. dExPath solved many problems by offering multi-round immunostaining, maximized protein epitope (another name for antigens) preservation, and elimination of fluorescent overlaps. Immunostaining normal brain tissue and brain tumor tissue has helped identify numerous improvements and advantages to dExPath. (Valdes et al., 2021)

dExPath is a form of microscopy technology that facilitates the immunostaining of cells, so far only applicable to the brain and brain stem cells. Immunostaining utilizes antibodies and conjugated enzymes to generate fluorescence as an indicator of certain antigens and markers on cells of interest. At its core, dExPath is a form of expansion microscopy, a preparation and imaging technique used to visualize biological nanostructures. Wassie et aldescribed the process as synthesizing a dense and even network of swellable polyelectrolyte hydrogel to react with specific nanostructures and physically expand them prior to imaging. Once expanded, immunostaining agents were able to label antigens of interest on cells, all of which were visible from an ordinary laboratory microscope.

Experimental uses of dExPath have allowed researchers to detect distinguishing epitopes and their combinations on glial cell tumors, offering a possibility for the technology’s use in detecting brain cancers. dExPath also facilitated multi-round immunostaining, where multiple forms of staining were implemented to highlight different structures in one image, eliminating the need to run multiple images for each layer of staining.

dExPath has solved many issues that persisted in ExPath. The predecessor failed to preserve some binding sites on protein epitopes, leaving a number of antigens unaccounted for before the use of dExPath. During imaging, the new technology also addressed the prior issue of fluorescent overlaps, particularly due to the presence of lipofuscin, a common waste product among cells. Lipofuscin is often mistaken for a different cellular marker of interest, but decrowding allowed for improved access of antibodies to epitopes, allowing the imaging technology to more clearly recognize its intended targets and ignore the lipofuscin autofluorescence. dExPath’s capacity outperformed Sudan Black B, a stain that reduced the autofluorescence of lipofuscin but weakened other relevant fluorescent signals.

Valdes et al. (2021) used dExPath to expand and investigate human and animal brain cell samples. Samples of normal, low-grade (slow-growth) tumors and high-grade (faster-growth) tumors were examined before and after the expansion process to detect differences in marker visibilities. The samples were immunostained for markers vimetin, Iba1, and GFAP. The presence and frequency of particular markers helped convey new indicators for stages of malignant growth in brain tissues. For example, GFAP and vimentin-labeled low-grade cells were interpreted as an indication of potential future malignancy. Additionally, cells labeled with Iba1 and GFAP indicated a possible increase in tumors with phagocytic (ingestive) properties that made them more invasive.

Figure 2, Immunostaining examples of vimetin, Iba1, and GFAP staining via. dExPath. Row 1 exhibits pre-expansion staining. Rows 2 & 3 exhibit pre-decrowding staining, and rows 4 & 5 exhibit post post-decrowding staining. Pre and post-expansion staining alternate starting from row 2. (Valdes et al., adapted from Figure 7)

Fluorescence imaged by dExPath is of significantly higher quality and consistency. dExPath revealed that there are significantly more cells of interest, with many corresponding to more different classes of cells, such as macrophages and glioma cells, that are deemed important to glioma pathology than previously expected.

dExPath is already demonstrating tremendous potential in brain disease analysis and diagnosis, as well as accuracy and reliability to an unprecedented degree. The technology’s compatibility with low-cost, conventional microscopes and more commercially available reagents and instruments in basic laboratories makes the technology tremendously accessible and convenient. Its advantages uncovered features after molecular expansion that uncovered cell demographics previously unaccounted for and redefined the aggressive nature of diseased cells. However, there are many more tests that dExPath must undergo, such as assessing its visualization of other neurological diseases or cancers. Continued research concerning dExPath may allow the world of science and medicine to uncover more about mysteries in neurobiology, and possibly introduce dExPath to clinical settings as a new assessment tool for neuropathology diagnosis.

Sources

Valdes, Pablo A., et al. Decrowding Expansion Pathology: Unmasking Previously Invisible Nanostructures and Cells in Intact Human Brain Pathology Specimens. 7 Dec. 2021,

Wassie, Asmamaw T., et al. “Expansion Microscopy: Principles and Uses in Biological Research.” Nature Methods, vol. 16, no. 1, 2019, pp. 33–41, https://doi.org/10.1038/s41592-018-0219-4.

Distributions, not Differentiation: New Studies Find Cell Proportions at the Heart of Neurological Specialization

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The human brain is organized into cortices, lobes, hemispheres, and more, with every designation serving as a location where a particular function necessary for survival is hosted. In understanding the cell types of the brain, scientists can further shape the understanding of the nature of human life. Current work strives toward comprehending the functions and capacities of the brain and developing stronger foundations for modeling brain physiology to support future research and medical applications. All of the advancements discussed originated from Alyssa Weninger and Paola Arlotta’s Science review article, A family portrait of human brain cells, which compiles recent findings in brain mapping research as aligned with the National Institute of Health’s BRAIN Initiative. In the article, Weninger and Arlotta summarize and discuss the work of multiple groups of neuroscientists that have developed new findings about the brain’s composition and variability across regions, individuals, and species (specifically five primates of interest and mice).

As suggested by the article, recent research from multiple teams of neuroscientists utilized a variety of study mechanisms to compare the composition of the brain. One of the most important tools used in the studies included single-cell profiling. This profiling technique analyzes cellular behavior through multiple methods that include their transcriptome (range of genetic information produced to control cell behavior), proteome (range of proteins produced by the cell), and epigenome (range of modifications and markings that control the genetic information expressed by a cell) to organize them into groups based on their functional similarities. Models that encompass these methods and human organoids (structures of organs derived from STEM cells that mimic organ tissue) are developed to model the brain and its cells. They are also used in mapping and developing comparative analyses to determine significant findings and understanding of the brain organization.

Comparisons of cell composition in regions across the brain resulted in findings from researchers under Siletti from the University of North Carolina at Chapel Hill and Jorstad from Harvard University. The two groups found that rather than mainly having different types of cells in different parts of the brain, some different parts of the brain shared the same cells but had different proportions of these cells. There were some exceptions, such as inhibitory neurons in the primary visual cortex, although the explanation of this finding is unclear. Such results change the understanding of evolutionary diversity in that diversification does not depend heavily on having many different cell types, but rather on having varying proportions of cells with small differences.

Jorstad’s group also developed a significant result in identifying differences in brain composition between human individuals. One cell type from 75 individuals was profiled and resulted in different classes of cells bearing contrasting levels of variability among individuals. Most of the explanatory factors were beyond demographic differences, such as gender, ancestry, or age. The reason for such differences is still unclear. Scientists are further encouraged to study bigger cohorts of people to further examine the origin of differences in variability across humans.

The finding of varying cell proportions held as Jorstad’s group conducted interspecies comparisons, comparing human compositions with other primates (specifically chimpanzees, gorillas, rhesus macaques, and marmosets). The exceptional cognitive ability found in humans was largely supported by differences in proportions of brain cell types rather than the variability of cell types. Additionally, faster evolutionary divergence may explain the differences in gene expression found between supportive tissue, known as glial cells, in the brain. This allowed for further species-specific development across primates. Only a limited number of gene patterns specific to humans were found, most of them concentrated in parts of the brain with human evolutionary change. As such, scientists have come to understand that attributes of the human brain are derived from very few cellular or molecular changes, leaving differences in cell proportions as the most prominent explanatory factory for human brain development. Furthermore, understanding the brains of related primates and their relation to human brains will help scientists develop new models for brain pathways and understand the kinds of questions that they will be able to answer with such knowledge in the future.

Neuroscientists today continue to work hard toward developing human brain models. Current studies are focusing on developing accurate organoids – three-dimensional tissue models of stem cells developed to mimic organs in structure and function. Velmeshev’s group of researchers worked towards profiling different cortical (outer layers of the upper brain) areas and related areas in fetuses to track developments across human births. Kim’s group of researchers investigated single-cell transcriptomes of the thalamus (the processor of sensory data) during its development but was missing an investigation of the thalamus cellular compositions. The work of these scientists contributes to the idea of molecular mechanisms as the driver of cellular diversity in the brain, but also calls for more innovation in external biological investigations to better model the brain and further study its composition. In doing so, neuroscientists will come even closer to understanding one of the most complex systems in the human body and develop more answers for current-day neurological problems.

Bibliography

Weninger, Alyssa, and Paola Arlotta. “A Family Portrait of Human Brain Cells.” Science, vol. 382, no. 6667, Oct. 2023, pp. 168–69, https://doi.org/10.1126/science.adk4857.