Functional Tissue Units in the Human Reference Atlas

Functional tissue units (FTUs) form the basic building blocks of organs and are important for understanding and modeling the healthy physiological function of the organ and changes during disease states. In this first comprehensive catalog of FTUs, we document the definition, physical dimensions, vasculature, and cellular composition of 22 anatomically correct, nested functional tissue units (FTUs) in 10 healthy human organs. The catalog includes datasets, illustrations, an interactive online FTU explorer, and a large printable poster. All data and code are freely available. This is part of a larger ongoing international effort to construct a Human Reference Atlas (HRA) of all cells in the human body.


Introduction
The Human Reference Atlas (HRA) is an international effort of 17 consortia to build a freely available map of the healthy adult human body down to the single-cell level.At the highest level are organ systems, whose individual organs contain various tissues, also called anatomical structures.Tissues, in turn, consist of repeating structures known as functional tissue units (FTUs).Bernard de Bono et al. define FTUs as "a three-dimensional block of cells centered around a capillary, such that each cell in this block is within diffusion distance from any other cell in the same block" 1 .The description of the cells within an FTU and the anatomical location of the FTU form a so-called primary tissue motif (PTM).An FTU must support both metabolism and communication between cells.The maximum diffusion distance of oxygen and other molecules constrains the size of an FTU.Therefore, tissues and organs in the body consist of many repeating FTUs to perform the overall function of the organ.We previously generalized this original definition by describing an FTU as "the smallest tissue organization that performs a unique physiologic function and is replicated multiple times in a whole organ" 2 .Here, we extend the definition of FTUs by allowing them to form a nested hierarchy to accomplish various functions.In this perspective, we present a catalog of the first 22 FTUs for the HRA.The current version of this atlas contains (1) the length and diameter of each FTU; (2) the vasculature pathways that connect FTUs to the heart and amongst each other; (3) the cell types within each FTU; and (4) 2D illustrations of each FTU showing their prototypical cell types, cell shapes, sizes, and spatial arrangement.We map all anatomical structures to their corresponding Uber-anatomy Ontology (UBERON) ID 3 and all cell types to their Cell Ontology (CL) ID 4 to enable linkage to other datasets.We reference supporting scholarly paper evidence where applicable.We make the HRA's catalog of FTUs available as machine-readable downloadable files, through an HRA Interactive FTU Explorer website, and as a large printable poster.Much of the information we present here about FTUs exists in the literature but siloed by organ or tissue type and with no unifying conceptual or data schema framework.The creation of a catalog of FTUs as an integral part of the HRA, along with standardized descriptions linked to ontologies, makes it possible to compare and contrast these units throughout the human body.This way, we can gain a better comprehension of how FTUs function together, and how they can be integrated into models and simulations.

HRA FTU Datasets
This initial release of the atlas contains 22 FTUs across 10 organs, see    1 includes the UBERON ID, dimensions, and references for each FTU.HRA FTUs are developed in a five-step process: 1) identify FTU shape, dimensions, and cell types from experimental data published in scholarly papers; 2) invite organ experts to comment on these and revise as needed; 3) a professional medical illustrator creates a vector-based drawing of the FTU and saves it as an SVG file with all metadata; 4) organ experts review the drawings and any existing disclaimers and suggest changes as needed; 5) number of cells per cell type are recorded and the FTU file is published with all metadata together with a crosswalk file as part of an HRA release.Subsequently, we detail key data types and formats.

Geometric Properties
Table 1 lists the 10 organs and their 22 FTUs together with the UBERON ID, spatial dimensions, and reference paper(s) in which the dimensions were published.For circularshaped FTUs, such as the alveolus of the lung, a single dimension value (or range) is listed representing the typical diameter of the FTU.For cylindrical FTUs, such as the inner medullary collecting duct in the kidney, both length and diameter are provided.The diameter of the intercalated duct is the largest at the head of the pancreas and smallest in the tail.

Vasculature
The HRA-VCCF dataset contains a list of all the blood vessels in the HRA, along with their branching structure, cell types, biomarkers, and other information 5,6 .Table 2 lists the vessels in the HRA-VCCF that directly supply or drain each FTU.Note that the epidermal ridge does not contain blood vessels, but rather obtains oxygen via diffusion from the underlying dermal papilla.Supplemental Table S1 lists the full vasculature pathways from the heart to each FTU and back to the heart.Where possible, vessels are mapped to their corresponding UBERON ID or Foundational Model of Anatomy (FMA) ontology IDs 7 .About 63% of the vessels exist in one or both of these ontologies.

Cell Types
A list of the different types of cells in each FTU is available at https://humanatlas.io/assets/table-data/ftu-cell-count-5th-release.csv.Table 3 shows a subset of this list for FTUs within the kidney nephron.Each cell type is associated with its corresponding Cell Ontology (CL) ID.Ongoing research using methods such as single-cell RNA sequencing is identifying biomarkers expressed in these cell types and determining the relative distribution of the cell types in the FTUs.Examples for the liver 8 and lung 9 are available at https://www.ebi.ac.uk/gxa/sc/experiments/E-MTAB-10553/results/anatomogram and https://www.ebi.ac.uk/gxa/sc/experiments/E-GEOD-130148/results/anatomogram.

Spatial Arrangement of Cells
The spatial arrangement of cells and their types in each FTU is recorded in 2D illustrations, see https://humanatlas.io/2d-ftu-illustrations.Medical illustrators create these illustrations by compiling information from papers, histology, and microscopical images of the FTU guided by Standard Operating Procedures (SOP) 3 and the Style Guide for Human Reference Atlas 2D Functional Tissue Unit (FTU) Illustrations 10 .The information is validated by Subject Matter Experts (SMEs) with extensive expertise in human anatomy and single-cell studies.The illustrations are saved in SVG format, and converted to JSON files using a code that helps to map the drawings to the metadata; and a Crosswalk table is compiled that associates 2D anatomical structures and cell types in the FTUs with their proper terms in the HRA.Illustrations of all 22 FTUs are shown in Fig. 1 at four levels of magnification, see scale bars.

HRA FTU Visualizations
In addition to accessing the HRA FTUs through machine-readable data files, users can also explore the data online using the Interactive FTU Explorer or as a printable poster.

Interactive FTU Explorer
Prior work by the European Bioinformatics Institute (EBI) on interactive anatomograms 11,12 and by the Kidney Precision Medicine Project (KPMP) on the Kidney Tissue Atlas Explorer 13 inspired the design of the lung and kidney FTU illustrations (creating de-facto standards across these three sites) and the design and implementation of the HRA Interactive FTU Explorer at https://hubmapconsortium.github.io/hra-ui/apps/ftu-ui/.As illustrated in Fig. 2, the website lets users select any of the 22 FTUs on the left, which brings up an interactive rendering of the FTU illustration in the middle.Cell types and their biomarkers derived from experimental data are tabulated in the top right; with circle size indicating the percentage of cells in the FTU and circle color representing the mean expression value over all cells of this type in the FTU-averaged over all datasets loaded for this FTU.Users can choose to explore gene, protein, and lipid biomarkers for different cell types using three tabs; in some cases, experimental data might not (yet) exist.In the lower right of the FTU Explorer, users can click on a specific source dataset and go to the portal that serves this dataset.In the next iteration of the FTU Explorer, users will be able to select specific datasets (e.g., datasets from different demographics) to understand changes in cell type by biomarker expression values as we age (young vs. old) or by sex (male vs. female).

Printable Poster
To place the FTUs within the larger context of the HRA, we created a printable poster visualizing all 1,607 anatomical structures and 1,943 cell types available in the 5th release of the HRA.The visualization is composed of two radial tree graphs: (1) The first graph contains the nested "partonomy" of the anatomical structures and cell types in the HRA.The human body serves as the root node in the center, the largest anatomical structures (organs) are placed further out, and smaller sub-structures and tissue types of branch outwards from the organs' leaf nodes denoting cell types.(2) The second graph contains all the blood vessels in the HRA, with the chambers of the heart in the center, and increasing smaller vessels more distal to the heart again branching outwards from the center.Nodes in the two radial tree graphs meet at points where the HRA indicates a vessel supplies or drains the corresponding anatomical structure.Nodes that represent FTUs are highlighted in green.The left side shows the nested partonomy and blood vessels in females and the right side shows them for males with a "butterfly-like" appearance that invites closer examination, discussion, and self-portrait photos, see Fig. 3.The 6-foot diameter poster is available in ready-to-print formats via https://github.com/cns-iu/hra-vccf-ftu-supporting-information.Note that there are 54 nodes for the 22 FTUs.This is because some FTUs overlap multiple anatomical structures and therefore appear in more than one location of the partonomy.We plan to update this poster with future releases of the HRA to reflect ongoing development of the partonomy and vasculature graphs and new FTUs added to the catalog.

Discussion
FTUs are the basic building blocks of organs.Their distinctive size (relative to diffusion distances) and physical arrangement of different cell types are key to enabling their corresponding physiologic function.In this perspective, we described datasets and visualizations of 22 FTUs in 10 organs interconnected by vasculature as published in the 5th release of the HRA.To our knowledge, this is the first time that comprehensive data specifically about FTUs across the human body have been systematically cataloged.At the macroanatomical scale, the vascular pathways connecting FTUs to the heart and each other, along with our HRA poster, show the position of FTUs in the body.At the microscopic scale, the illustrations and cell type table show the internal structure of the FTUs.Within the HRA effort, we are using this catalog of FTUs for several use cases.For example, we ran Kaggle competitions to develop scalable and generalizable segmentation code to identify FTUs in images 2,14 .We use the segmentation code to automatically count the number of FTUs per unit area and to compute general biomarker expression values for genes, proteins, lipids, and metabolites.Ongoing research compares hierarchical cell neighborhoods 15 computed from tissue data with the 22 FTUs in the catalog presented here.More broadly, we envision the FTU catalog and the Interactive FTU Explorer as a framework for researchers to study how the information presented here (FTU dimensions, cell types, etc.) varies across donor demographics (e.g., age, sex, race) and in different disease states.Understanding similarities and differences between FTUs can help predict adverse events of medications or suggest new drug targets.The FTU Explorer can be used as an educational tool, especially for comparing the physiology and vasculature of different organs and tissues.Our downloadable poster is intended for general public outreach.A limitation of this work is that the HRA is still in development, with updates planned every six months for the coming three years.As a result, this is not yet a complete map of all FTUs in the body, but rather a starting point demonstrating how various types of data about FTUs, including physical dimensions, vasculature, cell types, and spatial orientation can be interlinked across scales, in support of creating a human reference atlas.Tables Table 1.Functional tissue units by organ, with UBERON ID, and dimensions (length where applicable and diameter in millimeters) as listed in the provided references.

Figures Figure 1 .
Figures Figure 1.2D illustrations of all 22 FTU with name and size annotations.

Figure 2 :
Figure 2: HRA Interactive Functional Tissue Unit Explorer showing the 2D illustration of the renal corpuscle of the kidney along with associated experimental data.

Table 2 .
Blood vessels that directly supply or drain each FTU.

Table 3 .
Cell types with CL IDs in kidney nephron FTUs.

Table S1 .
Vascular pathways from the heart to each FTU and back to the heart https://github.com/cns-iu/hra-vccf-ftu-supporting-information/tree/main/data