Role of N-Cadherin in the Regulation of Hematopoietic Stem Cells in the Fetal Liver and Bone Marrow
Authors: Toyama Hirofumi , Hosokawa Kentaro , Ikushima Yoshiko Matsumoto , Suda Toshio , Arai Fumio 
Published: October 2019

Abstract
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.

Disclosures:
No relevant conflicts of interest to declare.