Interrogating the Spatial Architecture of Human Bone Marrow Via Imaging Mass Cytometry
Authors: Oetjen Karolyn A. , Bender Diane E. , Ruzinova Marianna B. , Oh Stephen T. , Link Daniel C. 
Published: November 2019

Hematopoietic stem cell (HSC) proliferation, self-renewal, differentiation, and trafficking are dependent, in part, upon signals generated by stromal cells in the bone marrow. Stromal cells are organized into niches that support specific subsets of hematopoietic progenitors. Intimate interactions between HSCs and neighboring stromal cells coordinate hematopoietic responses during periods of physiologic stress, while also maintaining the lifelong integrity of the hematopoietic stem cell pool. Hematopoietic niches are comprised of a heterogeneous population of stromal and hematopoietic cells. The identity and function of the known stromal cell subsets have primarily been gleaned through genetic manipulation of mouse models. In humans, the spatial organization of these stromal cells in bone marrow and the signals they generate to regulate hematopoiesis are poorly understood. Current methods to characterize bone marrow mesenchymal stromal cells in humans include: 1) immunostaining of bone sections; 2) analysis of flow sorted stromal cells; and 3) analysis of ex vivoexpanded mesenchymal stem/progenitors. Major limitations to all of these approaches exist that relate to the heterogeneity of bone marrow stromal cells, the lack of markers that reliably distinguish different stromal cell populations, and inherent technical limitations of the assays, such as the number of markers that can be analyzed at one time. Here we report our efforts to use imaging mass cytometry imaging (IMC) to interrogate the complex cellular architecture of human bone marrow.
IMC allows for the simultaneous detection of up to 40 markers through the use of antibodies conjugated to elemental metal tags acquired by time-of-flight mass spectrometry. We first used standard immunostaining techniques to develop a panel of antibodies compatible with archived formalin-fixed paraffin-embedded (FFPE) human bone marrow specimens using a heat-induced epitope retrieval method. Hematopoietic lineages have been successfully identified using CD11b, CD68, CD15, CD14, CD16, CD11c, CD20, CD3, CD4, CD8a, CD38, CD45RA, CD45RO, CD235a, CD71, CD34, CD31; stromal cells and structures with CXCL12, alpha-smooth muscle actin, collagen I, vimentin; and nuclear staining using Ki67, Histone H3, and DNA intercalator. We used panels consisting of up to 18 of these antibodies each to test IMC on FFPE human bone marrow specimens. Imaging was performed using the Hyperion Imaging System (Fluidigm), which consists of a UV laser scanning module to capture regions of more than 10,000 cells with 1-micron resolution coupled with a Helios Mass Cytometer. Image analysis was performed by creation of a cell segmentation mask using CellProfiler software and high dimensional analysis in histoCAT. Representative images and corresponding dimensional reduction with t-SNE are shown in Figure 1A-H, demonstrating successful discrimination of distinct hematopoietic lineages. Gating on subpopulations within the t-SNE clusters can be projected on to the original image to illustrate spatial distribution and marker co-expression, as an example CD3+ and CD8+ cells are shown in Figure 1I.
These data indicate that IMC allows for highly multiplexed analysis of bone marrow cell populations. Developing imaging techniques for analysis of tissue-banked FFPE bone marrow samples would have broad applications for translational research on hematologic diseases. In particular, this technology has tremendous potential to advance understanding of the spatial architecture of human bone marrow and to investigate alterations in the bone marrow environment in malignant hematopoiesis.

Disclosures
Oh: Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.