High resolution 3D mapping of cellular heterogeneity within multiple types of mineralized tissues

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Overall Abstract: The goal of the HuBMAP program is to appreciate the unique contextual role of individual cells within the 3D structure of a tissue at its most basic level of transcriptional activity, cellular signaling and cellular response. To date the subject tissues have not included the mineralized skeletal system due to technical issues that preclude the requirements of the HuBMAP program. We have solved those issues with a protocol that is capable of performing multimodal histology that include methods for advanced and repetitive in situ hybridization. We will develop this technology for three very different mineralized skeletal tissues: tooth, trabecular bone and cartilage structures of the knee. The Coordination Core will acquire the three tissue types from de-identified human sources. Each sample will be oriented to its source tissue, imaged by µCT to capture its mineral structure and processed into a histological stack to create a 3D representation of the tissue. Using the histological stack, the Mineralized Tissue Program will perform both in situ hybridization to identify the multiple cell types and seqFISH hybridization to capture the cell transcriptome of the identified cell types. These technically demanding steps will require direction from established HuBMAP investigators as well as UConn faculty who are experts in high resolution confocal microscopy. The Data Analysis Core will translate the image files generated by these techniques into 3D cellular maps of the target tissue and transcriptome composition of each cell type. From those data files, our contextual molecular mapping program, TOPAS, will examine the transcriptional and signaling pathways to impute how neighboring cells coordinate their activities to respond to mechanical loading and systemic factors that regulate skeletal health. The workflow and analytical platforms that we developed for the skeletal system will be aligned with the requirement of the HIVE including an outreach initiative. First, we will provide opportunities to transfer the histological technology to major academic skeletal research groups. Second, once our skeletal data becomes available from the central HIVE source, we will develop virtual workshops to inform the skeletal biology community of this valuable resource and how it can be utilized to unravel rare diseases affecting the skeleton. HuBMAP will be a transformational technology that every tissue centric group needs to incorporate. Our role is to ensure that the skeletal biology community is included in this new experimental platform, and that it is employed to solve the major genetic and therapeutic challenges affecting skeletal health.