A novel inducible haematopoietic cell‐depleting mouse model for chimeric complementation of blood cells

Abstract Haematopoietic stem cell transplantation (HSCT) is widely used in regenerative medicine. HSCT can be used not only to treat certain types of blood cancer and immune disorders but also to induce immune tolerance in organ transplantation. However, the inadequacy of HSCs available for transplantation is still a major hurdle for clinical applications. Here, we established a novel inducible haematopoietic cell‐depleting mouse model and tested the feasibility of using chimeric complementation to regenerate HSCs and their progeny cells. Large populations of syngeneic and major histocompatibility‐mismatched haematopoietic cells were successfully regenerated by this model. The stable allogeneic chimeric mice maintained a substantial population of donor HSCs and Tregs, which indicated that the donor allogeneic HSCs successfully repopulated the recipient blood system, and the regenerated donor Tregs played essential roles in establishing immune tolerance in the allogeneic recipients. In addition, rat blood cells were detected in this model after xenotransplantation of rat whole bone marrow (BM) or Lin− BM cells. This mouse model holds promise for regenerating xenogeneic blood cells, including human haematopoietic cells.

mouse model and tested the feasibility of using chimeric complementation to regenerate HSCs and their progeny cells. Large populations of syngeneic and major histocompatibility-mismatched haematopoietic cells were successfully regenerated by this model. The stable allogeneic chimeric mice maintained a substantial population of donor HSCs and Tregs, which indicated that the donor allogeneic HSCs successfully repopulated the recipient blood system, and the regenerated donor Tregs played essential roles in establishing immune tolerance in the allogeneic recipients. In addition, rat blood cells were detected in this model after xenotransplantation of rat whole bone marrow (BM) or Lin À BM cells. This mouse model holds promise for regenerating xenogeneic blood cells, including human haematopoietic cells.

| INTRODUCTION
Organ transplantation is widely used for the treatment of patients with end-stage organ failure. However, the shortage of organs available for transplantation makes it hard to meet the existing demands, 1 and many patients die while waiting for a transplantable organ. 2 Although HLA matching between organs of donors and recipients is performed during transplantation, most transplanted recipients have to persevere with lifelong immunosuppressant drug regimens, which might result in a risk of life-threatening infections. [3][4][5][6][7][8] Haematopoietic stem cell transplantation (HSCT) has been successfully used to induce tolerance during organ transplantation by the establishment of mixed haematopoietic chimerism. In addition, HSCT has been widely applied to the treatment of certain types of blood cancer and immune disorders. [9][10][11][12] Currently, tens of thousands of patients are cured through HSCT every year. However, the shortage of HSCs available for Weiyun Cao and Jiani Cao contributed equally to this study. transplantation is still a major hurdle for this therapy. Acquiring HSCs by differentiation of pluripotent stem cells has been suggested as a promising strategy to solve this problem. Encouraging results have been obtained from HSC differentiation in vitro; however, the difficulties in functional maturation of terminally differentiated cells and low differentiation efficiency still hinder this strategy. 13,14 The Vav1 gene has been reported to be expressed in virtually all haematopoietic cell lines but in very few other cell types. [15][16][17][18][19][20] In this study, we have developed an inducible haematopoietic cell-depleting mouse model by introducing the Vav1-HSVtk suicide cassette into the mouse genome. This cassette expresses the herpes simplex virus (HSV) thymidine kinase (tk) gene in haematopoietic cells under control of a Vav1 regulatory element. In the presence of ganciclovir (GCV), the HSVtk protein generates a toxic derivative, which induces apoptosis in cells. By using this mouse model, we have successfully regenerated syngeneic, allogeneic, and xenogeneic blood cells by bone marrow (BM) transplantation. This strategy holds promise for regenerating xenogeneic blood cells, including human blood cells, for regenerative medicine.

| Animals
All animal studies were performed by the principles approved by the

| CCK-8 assay
The cells were incubated with 20 μL enhanced Cell Counting Kit-8 (CCK8) solution per well for 4 h at 37 C. Then the OD values at 450 nm and 630 nm were evaluated by a microplate reader (Bio Tek).

| Western blot
Spleen T cells were harvested and lysed with RIPA buffer and protein concentrations were determined using a BCA assay. Equal amounts of proteins were then loaded on 12% SDS polyacrylamide gels. The separated samples were transferred to a PVDF membrane and incubated with the primary antibodies against the target proteins and then with the HRP-conjugated secondary antibodies. The protein bands were visualised by enhanced chemiluminescence using a bioimaging analyser. The proteins were detected using antibodies against β-actin (diluted 1:2000) and cleaved-caspase3 (diluted 1:1000). β-Actin was used as a loading control.

| Flow cytometry analysis
The samples were incubated with red blood cell lysis buffer for 10 min at 4 C, then incubated with the appropriate antibodies (Table 1)    To generate syngeneic, allogeneic, and xenogeneic haematopoietic chimeras, the donor cells were injected into the tail veins of recipient mice, and GCV (100 mg/kg) was administered to HBR mice via intraperitoneal (i.p.) injection from day À7 to day 7 daily and every other day for the next 3 weeks.

| Generation of a mouse model with inducible depletion of haematopoietic cells
To test the feasibility of regenerating blood cells using chimeric complementation, we generated a transgenic (Tg) mouse model with inducible depletion of the haematological system, in which the expression of the suicide gene HSVtk is driven by the murine Vav1 gene regulatory element HS21/45. In this model, the haematopoietic cells can be induced into apoptosis by injection of GCV ( Figure 1A). As expected, the spleen cells isolated from this mouse were killed by GCV treatments in a dose-dependent manner ( Figure 1B). Accordingly, the level of cleaved caspase3 protein in HSVtk Tg spleen T cells was up-regulated by increasing doses of GCV ( Figure 1C).
Next, we performed a titration assay to determine the safe dosages for GCV treatment in vivo ( Figures S1 and S2). Wild-type (WT) mice were continuously treated with GCV at dosages of 0, 20, 40, 80, or 160 mg/kg for 15 days. Cell apoptosis and mouse body weight were measured every 3 days during the titration period, and tissue histology was evaluated at the end of the titration assay ( Figure S1A). The results showed that there were no significant differences in cell apoptosis between the GCV treatment groups and the control groups at the tested dosages ( Figure S1B); however, the body weight of the 160 mg/kg group was significantly reduced compared with the control group ( Figure S1C). The histological data indicated that the cortex and medulla of the thymus tissue were dramatically  98.4% ± 0.1% (CD45.2 + B220 + ), and 90.7% ± 0.8% (CD45.2 + CD11b + Gr1/Mac1 + ), respectively ( Figures S3B and S3C). The BM cells from one HSVtk Tg mouse were able to generate 10 HSVtk-BM recipient (HBR) mice (designated HBR mice), which were used in the subsequent blood chimerism assays.

| GCV-induced haematopoietic cell depletion can be successfully compensated by syngeneic BMT
To test the feasibility of using HBR mice for chimeric complementa- of the GCV group, significantly higher than that of the PBS group ( Figure S4B). 80% of the mice in the GCV group survived at the 16th week after BMT, while the survival rate of the PBS group was 100% ( Figure S4C). Together, these data indicated that the transplanted CD45.1 haematopoietic cells, most likely including HSCs, are able to survive and repopulate the BM of recipient HBR mice.

| Allogeneic haematopoietic chimeras exhibit a high substitution level of donor HSCs and Tregs
We next tested whether allogeneic haematopoietic cells can repopulate the recipients by transplanting BM cells of Balb/c mice (H2K d ) into GCV-treated HBR mice (H2K b ) ( Figure 3A). Anti-CD4, anti-CD8, and anti-NK1.1 neutralising antibodies were additionally injected into the recipient mice one day before BMT to enhance the depletion of recipient leukocytes ( Figure S5). The results showed that transplanted  Figures 3D and S6A). Similar results were observed in the BM, thymus, lymph nodes, liver, and spleen of the recipient mouse ( Figure S6D).
Haematopoietic stem cells (HSCs) hold the ability to reconstitute all blood cell types. HSCs are enriched in the Lin À Sca1 + c-kit + (LSK) cell population in mouse BM. 24 The proportion of donor-derived H2K d LSK cells was 46.9% in the GCV-treated HBR recipient mouse, whereas the H2K d LSK chimerism in the PBS control group was 3.6% ± 0.4% at the end of the 16th week after BMT (Figures S6B and 3E).
These data suggest that the donor-derived HSCs integrated into the recipient haematopoietic niche and participated in haematopoietic reconstitution in the HBR recipient mice.

| Regeneration of rat haematopoietic cells in HBR mice
To test the feasibility of using HBR mice to regenerate rat haematopoietic cells, we transplanted BM cells of inbred Fischer (F344) rats into GCV-treated HBR recipient mice ( Figure 4A) Together, these data indicate that HBR mice can be used to regenerate xenogeneic rat haematopoietic cells, although with low levels of chimerism.

| DISCUSSION
The HSVtk/GCV suicide system has been widely used in cancer gene therapy. [25][26][27][28][29][30] In this system, thymidine kinase phosphorylates intravenously administered GCV into a toxic compound that is Aplastic anaemia is a disease of BM failure, and the clinical diagnosis is based on pancytopenia. 31 Previous studies showed that aplastic anaemia mice without therapeutic treatment suffered a 100% mortality rate within 45 days. 32 Immune rejection is a major challenge during the construction of haematopoietic chimeras. In the allogeneic and xenogeneic chimeric F I G U R E 4 Xenogeneic haematopoietic chimeras are obtained under the HSVtk/ganciclovir (GCV) system after the transplantation of rat bone marrow cells into HSVtk-BM recipient (HBR) mice. (A) Schematic of the procedure for constructing the xenogeneic (rat-mouse) haematopoietic chimeras; 15 Â 10 6 rat (rCD45) whole bone marrow cells or 8 Â 10 5 rat (rCD45) Lineage À cells (Lin À : rCD45 + rCD3 À rCD45RA À rCD161 À rSIRPA À ) were transplanted into HBR mice (mCD45) at day 0. Neutralising antibodies (anti-CD8/CD4/NK1.1) were administered via intraperitoneal (i.p.) injection 1 day before BMT, and i.p. injection of GCV was performed daily from week À1 to week 1 and every other day from week 2 to week 4. assays, the survival rates of transplanted HBR mice were significantly lower than that of control groups, and several of the surviving mice had lost the donor haematopoietic cells at the late stages of the experiment, judged by periodical analysis of peripheral blood (Figures 3 and 4). Tregs play a key role in immune tolerance during organ transplantation and infusion of donor Tregs to the recipient has been used as a strategy to alleviate transplant rejection. [34][35][36][37] There are several different sources of Tregs, among which thymus-derived Tregs (nTregs) have a better immunosuppressive effect, especially in the formation of peripheral tolerance. 38 In the allogeneic experiment, high chimerism of donor-derived Tregs was detected in the thymus tissues of recipient mice at 16 weeks after BMT (Figures S4B and 3F).
These results are consistent with a high chimeric ratio of cells from differentiated haematopoietic lineages in HBR PBMCs ( Figures 2D   and 3D). Together, these data support the notion that donor derived Tregs play critical roles in tolerance induction.
In this study, we successfully obtained rat-mouse xenogeneic haematopoietic chimeras using HBR mice. The maximum proportion of haematopoietic chimerism reached 29.4% ( Figure 4B). The rat haematopoietic cells were maintained for 4 weeks in HBR mice when transplanting whole BM, and for 8 weeks when transplanting Lin À BM cells of F344 rats ( Figure 4B,D). These encouraging data suggest that this model can be expanded to human haematopoietic cells.
In conclusion, we have successfully developed an inducible haematopoietic cell-depleting mouse model, which can be used for the regeneration of allogeneic and xenogeneic haematopoietic cells.
Although a relatively high ratio of allogeneic haematopoietic chimerism was achieved, the xenografted rat haematopoietic cells showed a