Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system

Yong, Kylie Su Mei; Ng, Justin Han Jia; Her, Zhisheng; Hey, Ying Ying; Tan, Sue Yee; Tan, Wilson Wei Sheng; Irac, Sergio Erdal; Liu, Min; Chan, Xue Ying; Gunawan, Merry; Foo, Randy Jee Hiang; Low, Dolyce Hong Wen; Mendenhall, Ian Hewitt; Chionh, Yok Teng; Dutertre, Charles-Antoine; Chen, Qingfeng; Wang, Lin-Fa

To optimize the production of bat-mice, further studies were conducted to improve the transplantation protocol and define the minimum number of bat BM cells needed to reconstitute NSG mice [52] [53] [54] . As compared to adult mice, neonatal NSG are known to be better at establishing human cell repopulation in humanized mice 25, 55 . Bat BM cells were prepared and injected into sub-lethally irradiated NSG neonates at varying numbers of 5 × 10 3 , 1 × 10 4 , 5 × 10 4 or 1 × 10 5 cells. As shown in Fig. 3a , according to peripheral blood reconstitution levels at 40 weeks post-injection, the groups of 5 × 10 3 and 1 × 10 4 cells did not give rise to significant reconstitution, whereas a transfer of 5 × 10 4 or more cells resulted in engraftment. However, to achieve a robust establishment and maintenance of bat immune cells in NSG recipients, an initiating number of 1 × 10 5 bat BM cells was required to reach a reconstitution level of ~20% to 50% (Fig. 3a) , which was comparable to adult NSG mice receiving 1 × 10 6 BM cells (Fig. 1c) . Systemic reconstitution in various organs such as BM, liver and spleen were analyzed ( Supplementary Fig. 2 ). The bat cell chimerism levels in organs from groups of 5 × 10 3 , 1 × 10 4 and 5 × 10 4 cells remained low. In mice injected with 1 × 10 5 cells, the reconstitution levels was robust with variation in different organs: in BM, levels of bat chimerism was ~20% to 35%, with bat cell numbers ranging from ~2 × 10 6 to 3 × 10 6 (Fig. 3b) ; in liver, reconstitution levels could reach ~50% to 75% with bat cell numbers of ~1 × 10 6 to 2 × 10 6 (Fig. 3c) ; spleen had the highest reconstitution levels and cell numbers which were ~70% to 80% and ~20 × 10 6 to 40 × 10 6 respectively (Fig. 3d) . The dramatic increase in the number of bat cells within the organs compared to the initiating number of cells injected, demonstrated that there was a massive in vivo expansion of bat cells in NSG mice (Fig. 3b to d) . Overall proportions of immune subsets, such as monocytes, T/NK, B cells and DCs also varied between the peripheral blood, bone marrow, liver and spleen. In the peripheral blood, monocyte levels were ~40% to 70%, T/NK cells ~20% to 58%, B cells ~2% to 5% and DCs ~2% to 5% (Fig. 3e) . In the bone marrow, monocytes ~80% to 95%, T/NK cells ~2% to 5%, B cells ~2% to 5% and DCs ~2% to 5% (Fig. 3f) . Within the liver, the proportion of monocytes was ~60% to 62%, T/NK cells ~20% to 30%, B cells ~10% to 20% and DCs ~0% to 0.05% (Fig. 3g) . The spleen was mostly dominated by monocytes, ~90% to 95%, with T/NK, B cells and DCs standing at ~2% to 5%, ~1% to 3% and ~0.5% to 2% respectively (Fig. 3h) . Altogether, it is evident that neonatal mice, with an engraftment of a limited number of bat BM cells, were able to achieve considerable levels of chimerism with all major immune cells present, therefore, enabling the reliable generation of a large cohort of bat-mice. Using the optimised protocol, we envisage that 80 to 100 bat-mice could potentially be generated from the BM of a single E. spelaea bat.