HuBMAP Collaborations Conduct RNA/Protein Comparison in Kidney, Multi-Group Analysis of Common Heart Sample

HuBMAP Collaborations Conduct RNA/Protein Comparison in Kidney, Multi-Group Analysis of Common Heart Sample

April 12, 2021

This HuBMAP webinar presented two collaborations focused on very disparate goals in assembling fine resolution, 3D maps of the human body. The first focuses on using complementary analysis of human kidney tissue using RNA- and protein-based probes; the second is pursuing a wide-scale analysis of the same sample of human heart tissue by different groups using a spectrum of methods.

Complementary Analysis via DART-FISH and Immuno-SABER

An intensive project to probe RNA and protein markers to create a single-cell spatial map of the human kidney is the focus of a collaboration between the Washington University and UC San Diego HuBMAP Tissue Mapping Centers (TMCs) and the Harvard University Transformative Technology Development (TTD) project. Sanjay Jain of WUSTL, Michel Nofal of Harvard and Kian Kalhor of UCSD described the project’s goals and achievements to date. Their presentation focused on the collaborators’ use of two probes, Immuno-SABER and DART-FISH.

Immuno-SABER, Nofal said, works similarly to CODEX in that it uses DNA-conjugated antibodies to image protein targets in a multiplexed manner. It differs from CODEX in that the DNA signal is amplified by a concatemer, which binds to the DNA strand docked to the antibody and contains dozens of instances of a repeated sequence that is recognized by fluorophore-tagged imager oligos. This signal amplification increases both throughput and sensitivity, enabling imaging of low-abundance targets not visible with other approaches. Four protein targets are visualized in each imaging round, then imagers are washed away to erase the signal in a non-destructive manner such that the next group of protein targets can be visualized. The method has produced promising images containing spatial information of 10 protein targets simultaneously. Challenges to be overcome are a relatively high failure rate for potential probes—possibly related to over-conjugation of antibodies—and the necessity to develop a strategy to cross-validate protein localization signals in high throughput.

Kalhor described the group’s work with DART-FISH, which works by reverse transcribing mRNA in the sample, targeting it with padlock probes which are amplified into “DNA nanoballs.” The researchers first decode the resulting fluorescence signal spots to determine expression patterns cell-by-cell. Marker overlap with Immuno-SABER is promising, with overall results in line with known kidney physiology.

Major goals for the collaboration, Jain said, include developing more DART-FISH probe sets, antibodies for Immuno-SABER and samples for both technologies; improving comparison between them via adjacent-slice images; and reagent optimization.

Investigating a Common Tissue

A wide collaboration of HuBMAP Consortium centers reported on their project to perform a complementary investigation of common tissue samples via similar and different assays. Shin Lin of the University of Washington described the collaboration’s efforts to establish the reproducibility and replicability of results, as well as the complementary information provided by each group’s investigation.

The effort began with distribution of left-ventricular tissue from a single deceased donor to 14 HuBMAP sites across the country. Both fixed and flash-frozen tissues were sent. Assays performed by the collaborators included nine prior-free and seven probe-based methods detecting nucleic acids and proteins. To speed the research along, two published, high-quality, 10x single-nuclei RNA sequencing (scRNA-seq) datasets (PMID: 32403949, 32971526) were used to inform the project.

For the spatial assays, the work is focused on mapping the major cell types of the left ventricular myocardium. RNA-based assays will choose a range of biomarkers to probe--from sets formulated by expert consensus to others which are more data driven--depending on the probeset capacity of their assay. For antibody-based assays, antibodies to five proteins, previously mapped by the GE RTI, will be used.

Beyond displaying major cell types of left ventricular myocardium in histologic images, another major focus for this effort is to explore subtype patterning in cardiomyocytes. The plan is to first find coexpression of proteins previously found to exhibit mosaicism by Wang et al. (PMID: 29677652) to determine subtypes of cardiomyocytes. These protein results will then be linked to multiplexed RNA-based spatial assays and snRNAseq signals to identify functional differences among these subtypes. The initial protein-based portion of this aspect of the project has been organized among the HuBMAP components (Stanford TMC, UF TMC, and VU TMC) in a “divide and conquer” strategy. Future goals include distributing tissue from a new donor to provide data from a biological replicate well as analysis of samples through the full thickness of the heart tissue.