Friday, May 9, 2025
5/9/2025
Heterogeneity of bone marrow cavities shaping the hematopoietic microenvironment - ABSTRACT The BM stromal cells (BMSCs) are a key element of the hematopoietic stem cell (HSC) niche. However, the niche is a concept based on spatial localization, whereas the stromal network permeates throughout the entire BM space, calling into question whether all BMSCs are equally endowed with niche-supporting functions, or just a subset of a heterogeneous BMSC population. The central hypothesis of this project is that the stromal cells in different types of BM cavities are functionally distinct, based on our observation that BM cavities are heterogeneous with respect to their bone remodeling activities. We found cavities that are predominantly undergoing bone deposition, resorption, or a mixture of the two, and classified them as D-type, R-type or M- type, respectively. Remarkably, clonal expansion of HSCs both under steady state and after stimulation with cyclophosphamide and G-CSF is found almost exclusively in M-type cavities. Similarly, early expansion of acute myeloid leukemia (AML) cells is also restricted to M-type cavities, underscoring the need to examine the stromal cells in the context of individual BM cavities undergoing different stages of bone remodeling. Here we propose to profile BMSCs from distinct cavity types using Image-seq (Aim 1), a new technology developed in our laboratory for capturing BM cells directly from specific microanatomic locations under image guidance for single cell RNA sequencing (scRNA-seq). Combining Image-seq with imaging bone remodeling activity, we will determine if BMSCs residing in D-, M-, and R-type cavities differentially express genes encoding factors for niche support and lineage differentiation. In Am 2, we will test the hypothesis that different BM cavity types are established by distinct stromal progenitor clones. We propose to employ a CRISPR based barcoding model (CARLIN) to track the progeny of stromal progenitor clones in vivo. The combination of this clonal tracking strategy with image-seq will provide unique insights into whether restricted stromal clones influence the R, D, and M cavities in the BM at steady state and after inflammatory challenge. We will further test if regenerative potential is linked to a specific cavity type by transplanting stroma from individual (D/M/R) sources to osteoporotic recipients in a fracture healing model. In Aim 3, we will examine if the BMSCs are interconnected to form a 3D functional network. Our preliminary data show that at least a subset of BMSCs display spontaneous intracellular calcium transients under steady state, which increase in intensity and frequency and can turn into intercellular calcium waves (ICWs) upon stimulation. Moreover, the calcium activity is spatially heterogeneous, with much higher activity in M- and R-type cavities compared to D-type cavities. We will map the BMSC network activity and investigate the relationship between calcium signaling and stromal cell function. We will examine whether disruption of calcium signaling in BMSCs leads to an altered microenvironment for hematopoietic support. This multidisciplinary research project will advance our understanding of the BM stromal compartment: its heterogeneity, clonality, and network activity.
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