Role of N-Cadherin in the Regulation of Hematopoietic Stem Cells in the Fetal Liver and Bone Marrow

Abstract 1205

The interaction of stem cells with their supportive microenvironmental niche is critical for sustaining stem cell pools in tissues over long periods of time. Cell-cell and cell-extracellular matrix interactions between hematopoietic stem cells (HSCs) and their niches contribute to the maintenance of stem cell properties. We previously demonstrated that N-cadherin mediated cell adhesion plays a critical role in the HSC engraftment and slow cell division of HSCs. Furthermore, in vitro culture of HSCs with bone-derived osteoblasts that expressed high levels of N-cadherin enhanced the LTR activity of HSCs. However, the expression and function of N-cadherin in HSCs is still controversial. A major problem is that there have been no specific anti-N-cadherin antibodies (Abs) that can be used for the detection of N-cadherin on the surface of living cells.

To address this problem, we produced a new anti-N-cadherin Ab. For the production of anti-N-cadherin Abs, we used the phage display library and isolated recombinant Ab clones against mouse N-cadherin. After screening of the phage library and performing quality control ELISA with positive and negative control proteins, we found that three of the seven newly-developed Ab clones were suitable for FACS. FACS analysis with a new N-cadherin Ab showed that BM LSK cells expressed low levels of N-cadherin protein. Furthermore, we confirmed that the reactivity of the new N-cadherin Ab was significantly reduced in N-cadherin deficient LSK cells compared to the wild-type LSK cells. RT-PCR and Q-PCR analysis revealed significantly higher levels of N-cadherin mRNA in N-cadherin⁺ LSK cells compared with N-cadherin^– LSK cells. Next, we performed BMT assays with adult BM-derived N-cadherin⁺ and N-cadherin^– LSK cells isolated by using the new N-cadherin Ab, clone AbD13081, and found that N-cadherin⁺ LSK cells showed higher BM reconstitution compared with N-cadherin^– cells. Furthermore, one of our N-cadherin Ab clones, AbD13077, has neutralizing activity and the use of this clone in cell sorting reduces the LTR activity of N-cadherin⁺ LSK cells. These data suggested that adult BM HSCs express N-cadherin.

Next we examined the expression of N-cadherin in the fetal HSCs using a new N-cadherin Ab. We found that a large number (29.3 ± 2.6 %) of LSK cells in E12.5 fetal liver (FL) expressed N-cadherin. Interestingly, N-cadherin expression was drastically decreased in E15.5 and E18.5 FL LSK cells (13.2 ± 1.9 % in E15.5 and 16.5 ± 1.4 % in E18.5). Immunohistochemical staining revealed that N-cadherin⁺c-Kit⁺ cells/N-cadherin⁺EPCR⁺ hematopoietic cells adhered to Lyve-1⁺ endothelial cells in E12.5 FL. Consistent with FACS analysis, N-cadherin expression was decreased in E15.5 and E18.5 FL. In contrast, the expression of E-cadherin in hepatic cells was significantly upregulated in E15.5 and E18.5 FL. Next we analyzed the expression of the LT-HSC marker, EPCR in N-cadherin⁺ and N-cadherin⁻ LSK cells. We found that EPCR⁺ cells were enriched in the N-cadherin⁺ LSK fraction in E12.5 FL, while there was no significant difference in the frequency of EPCR⁺ cells between N-cadherin⁺ and N-cadherin⁻ LSK in E15.5 and E18.5 FL LSK cells.

Finally, we performed the BMT assay with E12.5, E15.5, and E18.5 FL-derived N-cadherin⁺ and N-cadherin^– LSK cells isolated by AbD13081. Similar to the BM N-cadherin⁺ LSK cells, E12.5 FL N-cadherin⁺ LSK cells showed higher LTR activity than N-cadherin^– LSK cells. Interestingly, the advantage of LTR in N-cadherin⁺ LSK cells was decreased in E15.5 and E18.5 FL compared to E12.5 N-cadherin⁺ LSK cells, although the reconstitution of the N-cadherin⁺ fraction was higher than N-cadherin^– fraction.

Altogether, these data suggest that N-cadherin is highly expressed in E12.5 FL HSCs and plays an important role in the HSC-niche interaction for the maintenance of HSC activity. We speculated that the decrease of N-cadherin in HSC and FL cells during FL development might contribute to the migration of HSCs from FL to BM.