CD166 Engagement Augments Mouse and Human Hematopoietic Stem and Progenitor Function Via Activation of Stem Cell-Associated and Cell Cycle Pathways

Functions of hematopoietic stem and progenitor cells are regulated by cellular signaling networks and by the cellular and non-cellular elements of the hematopoietic niche. CD166 is highly expressed on both human and murine hematopoietic stem cells (HSC) and on murine osteoblasts and has been identified as a critical regulator of their functions. CD166 engages in trans-homophilic interactions with other CD166 molecules. However how homophilic or heterophilic engagement of CD166 (with CD6) improves HSC function is unknown.

Since we described that CD166 is expressed on murine and human HSC and murine osteoblasts, we hypothesized that CD166-CD166 homophilic engagement is critical for the hematopoiesis enhancing activity (HEA) that is mediated by osteoblasts. CD166⁺LSK cells cultured for 7d on either WT osteoblasts or recombinant murine CD166 (rmCD166) showed increased colony forming units (CFU) compared to CD166^-LSK cells cultured identically (p<0.05). Moreover, after 7d, the frequency of Lin^-Sca1⁺ cells was higher in co-cultures containing rmCD166 and CD166⁺LSK than cultures initiated with CD166^-LSK (p<0.05).

Next, we determined the effect of loss of CD166 expression on progenitors and osteoblasts on hematopoietic functions using CD166 knockout mice (CD166^-/-). CD166^-/-LSK cells co-cultured with rmCD166 showed decreased CFU production compared to WT LSK cultured similarly (p<0.05). In addition, WT HSC cultured on CD166^-/-osteoblasts for 7d showed decreased fold change in CFU relative to WT HSC cultured over WT osteoblasts (p<0.05). In vivo transplantation studies to corroborate our in vitro results are ongoing. To determine if CD166-CD166 interactions also enhance hematopoietic functions of human HSC, we cultured CD34⁺CD166⁺ and CD34⁺CD166^- HSC isolated from cord blood with recombinant human CD166 (rhCD166). On d7, CD34⁺CD166⁺ HSC cultured with rhCD166 showed 30±4-fold increase in CFU production compared to d0, whereas CD34⁺CD166^- HSC had only 9±0.4-fold increase. Taken together, these data suggest that CD166-CD166 homophilic interactions enhance hematopoietic functions and loss of this homophilic interaction negatively impacts HSC.

To elucidate the underlying signaling mechanism of CD166-CD166 mediated HEA, we cultured WT or CD166^-/-SLAM LSK cells with rmCD166 for 20hr and performed single-cell (sc) RNA seq. We analyzed for differential gene expression (DEG) using LGMT model and identified 518 upregulated and 174 downregulated genes in CD166^-/-HSC compared to WT. Following pathway enrichment analysis of DEGs, we identified 148 canonical pathways enriched by the upregulated genes in CD166^-/-HSC, including cell cycle, translational regulation, and mitochondria-related signaling pathways. 268 pathways were impacted by the downregulated genes in CD166^-/-HSC, including oxidative stress response, and metabolism. Moreover, CXCR4 signaling, PDGF signaling and glucocorticoid receptor signaling pathways were also downregulated in CD166^-/-HSC.

A cell trajectory reflecting associations among cells revealed a single cluster of CD166^-/-HSC. CD166^-/-HSC are linked with low expression of stemness marker genes, and high expression of genes regulating cell cycle, oxidative phosphorylation, and glucose metabolism. In addition, ER-stress and oxidative stress responsive genes are overexpressed in CD166^-/-HSC. In CD166^-/-HSC, genes of all enriched pathways were highly connected in the co-expressed networks, which indicates that in HSC, the impact of loss of CD166 on cell cycle, metabolism, growth factors and stemness pathways is highly associated. Sixteen hub genes including Suclg1, Eif4a1, Cox4i2, Jak3, Runx3, and Cdk6 were identified in the co-expression network.

We next applied bi-clustering algorithm QUBIC to identify modules of co-upregulated genes to analyze the transcriptomic variations of DEG in CD166^-/-HSC and identified 39-gene co-upregulation modules forming one big block in CD166^-/-HSC and 35-gene modules forming three blocks in WT HSC indicating that modules closely corresponded to CD166's impact over different cell states rather than on individual pathways.

Overall, our studies suggest that homophilic CD166 interactions involving HSC are required for maintenance of essential pathways that sustain HSC function and progenitor cell production including stemness, mitochondrial function, metabolism, cell cycle and growth factor signaling.