UF Diabetes Institute Investigators To Generate A 3D Map of the Human Lymphatic System

Grant Title: A 3D Map of the Human Lymphatic System

Program Director:

Mark Atkinson, PhD


Mark Atkinson, PhD (Project 1 and Administrative Core)

Harry Nick, PhD (Organ Donor Core)

Todd Brusko, PhD (Project 2)

Michael Clare-Salzler, MD (Project 3)

Bernd Bodenmiller, PhD, University of Zurich (Data Analysis Core)


Clive Wasserfall, MS

Kevin Otto, PhD

Irina Kusmartseva, PhD

Steven Goldstein, MD

As one of five Tissue Mapping Centers for the newly established NIH Human BioMolecular Atlas Program (HuBMAP), the University of Florida Diabetes Institute (UFDI) https://diabetes.ufl.edu/ will pioneer a groundbreaking initiative to map the three-dimensional (3D) cellular localization as well as gene and protein expression from human spleen, lymph node, and thymus, which together constitute the lymphatic system. Tissues will be acquired from deceased organ donors and processed immediately upon arrival to the Organ Donor Core at the University of Florida. Our procurement, administration, and processing teams are on call 24/7/365 to facilitate consent and case follow-up, organ acceptance, and timely processing. Organ quality and normal morphology will be histologically confirmed by Dr. Goldstein, a board certified hematopathologist. A portion of each organ will be freshly dissociated and assessed by flow cytometry for extensive human immunophenotyping (HIP) of 38 cellular subsets with 149 phenotypic determinations. In addition, dissociated cell subsets will be isolated and profiled by single-cell RNA-seq using the 10x Genomics Platform. Cell-specific transcripts will be interrogated through introduction into an imaging analysis pipeline. The optical microscopy pipeline will feature expansion microscopy, small molecule fluorescence in situ hybridization (smFISH), and multiplexed immunofluorescence microscopy and will employ five imaging platforms with increasing resolution (i.e., light sheet, multiphoton, widefield, confocal, and stochastic optical reconstruction microscopy (STORM)). From each tissue (spleen, thymus, and lymph node), investigators will assess microanatomical features, protein localization, and gene expression from 2D serial tissue sections with orientation tracked by various fiducial markers. Imaged tissue sections will then be shipped to Dr. Bodenmiller (University of Zurich) for analysis by imaging mass cytometry (IMC), which enables simultaneous detection of over 50 protein and RNA analytes from small regions of interest within the tissue section. Finally, 2D image files will be assembled for 3D reconstruction in silico using the histoCAT 3D toolbox, developed by Dr. Bodenmiller’s lab. This effort will first focus on “normal” tissues unaffected by known or observed pathologies in order to form a baseline understanding of the 3D microanatomy from each human lymphatic organ. Hence, these findings are expected to eventually facilitate important studies of tissue-specific alterations in morphology, cellular localization and gene/protein expression as it pertains to various immune-mediated diseases including autoimmune diseases and certain cancers.