Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing

Our research group is trying to understand the cell composition and cell-specific transcriptional profiles of intermuscular adipose tissue or IMAT in humans. IMAT is actually an ectopic adipose depot residing between and around muscle fibers. Computed tomography and MRI have been able to quantify IMAT volume and have recently shown that it increases with both age and BMI, and is also associated with muscle degenerative diseases.

In vitro secretome studies have recently shown that IMAT secreted proteins have the ability to influence insulin sensitivity, inflammatory profiles, and also contractile protein expressions in human skeletal muscle. The limited amount of IMAT tissue collected during biopsies and the lipid laden nature of adipocytes make this tissue incompatible with tissue digestion and single cell RNA-Seq. Therefore, we wanted to develop a protocol that performed nuclei isolation on small amounts of tissue that could then be used in downstream single nuclei RNA-Seq.

This protocol will enable researchers to determine the compositional and cell-specific transcriptional alterations that occur in IMAT in different metabolic disease states and in response to interventions. We hope that this information will be crucial in developing new therapeutic strategies to help alleviate metabolic disease by either targeting IMAT directly or its secreted factors. The field severely lacks data on the biology of IMAT, its cellular origins, and communication with other cells, resident both within and outside of IMAT, as well as its molecular responses to lifestyle interventions, such as diet and exercise or pharmacotherapy.

All of these are key research questions for our group.