Hoxb5 reprogrammes murine multipotent blood progenitors into haematopoietic stem cell‐like cells

Abstract Objectives The expression of transcription factor Hoxb5 specifically marks the functional haematopoietic stem cells (HSC) in mice. However, our recent work demonstrated that ectopic expression of Hoxb5 exerted little effect on HSC but could convert B‐cell progenitors into functional T cells in vivo. Thus, cell type‐ and development stage‐specific roles of Hoxb5 in haematopoietic hierarchy await more extensive exploration. In this study, we aim to investigate the effect of Hoxb5 expression in multipotent blood progenitor cells. Materials and Methods A Mx1cre/RosaLSL‐Hoxb5‐EGFP/+ mouse model was used to evaluate the effect of Hoxb5 expression in blood multipotent progenitor cells (MPP). Golden standard serial transplantation experiments were used to test the long‐term haematopoiesis potential of Hoxb5‐expressing MPP. Single‐cell RNA‐seq analysis was used to characterize the gained molecular features of Hoxb5‐expressing MPP and to compare with the global transcriptome features of natural adult HSC and fetal liver HSC (FL HSC). Results Here, with a mouse strain engineered with conditional expression of Hoxb5, we unveiled that induced expression of Hoxb5 in MPP led to the generation of a de novo Sca1+c‐kit+CD11b+CD48+ (CD11b+CD48+SK) cell type, which can repopulate long‐term multilineage haematopoiesis in serial transplantations. RNA‐seq analysis showed that CD11b+CD48+SK cells exhibited acquired features of DNA replication and cell division. Conclusions Our current study uncovers that Hoxb5 can empower MPP with self‐renewal ability and indicates an alternative approach for generating HSC‐like cells in vivo from blood lineage cells.

showed that CD11b + CD48 + SK cells exhibited acquired features of DNA replication and cell division.
Conclusions: Our current study uncovers that Hoxb5 can empower MPP with selfrenewal ability and indicates an alternative approach for generating HSC-like cells in vivo from blood lineage cells.

| INTRODUCTION
Haematopoietic stem cell (HSC) is the blood cell type that possesses dual features of self-renewal and multilineage potential, which are critical for replenishing the entire haematopoietic system throughout an individual lifespan. 1,2 However, the absolute numbers of HSC in adults are extremely rare 3,4 and are not efficiently expanded in vitro. 5,6 Researchers have been attempting alternative approaches to generate engraftable blood progenitors by enforcing expressing those molecules highly expressed in HSC but absent in downstream progenies. Ectopic expression of Sox17 can confer self-renewal potential on adult haematopoietic progenitors. However, this approach eventually led to leukemogenesis. 7 Likewise, miR-125a is a non-coding RNA gene preferentially expressed in HSC rather than blood progenies. 8 Ectopic expression of miR-125a in mouse haematopoietic progenitors induced long-term haematopoiesis, but the recipient mice suffered an MPN-like disease after secondary transplantation. [9][10][11] Therefore, more extensive and innovative efforts are needed to develop safer approaches to convert blood progenitor cells into engraftable blood stem cells for ultimately therapeutic uses.
Hoxb5, a member of HOX gene family, is preferentially expressed in HSC and uniquely marks the long-term HSC. 12,13 Our recent study showed that the gain of function of Hoxb5 in pro-pre-B cells reprogrammed these cells into T lymphocytes in vivo. 14 Moreover, the latest research shows that exogeneous Hoxb5 expression confers protection against loss of self-renewal to Hoxb5-negative HSCs and can partially alter the cell fate of ST-HSCs to that of LT-HSCs. 15 Here, we further studied the potential role of Hoxb5 in the MPP cell context, an intermediate progeny of HSC without self-renewal ability. Interestingly, conditional overexpression of Hoxb5 in MPP upon transplantation led to long-term haematopoiesis in serially transplanted mice.
More importantly, Hoxb5 resulted in a de novo cell type defined as CD11b + CD48 + SK, which contributed to the sustainable long-term haematopoiesis in serially transplanted recipients. CD11b + CD48 + SK cells exhibited features related to DNA replication and cell division. This study reveals de novo evidence that Hoxb5 can efficiently reprogramme blood progenitors into engraftable blood stem cells.

| Mice
Animals were housed in the animal facility of the Guangzhou Institutes of Biomedicine and Health (GIBH). Rosa LSL-Hoxb5-EGFP/+ mice were described as previously reported. 14 Mice of the CD45.1 + and Mx1-cre strains were purchased from the Jackson laboratory. All the mouse lines were maintained on a pure C57BL/6 genetic background.
All experiments were conducted in accordance with experimental protocols approved by the Animal Ethics Committee of GIBH.

| Cell sorting
Bone marrow cells used for transplantation or RNA-seq were first incubated with the biotin-conjugated antibody to Sca1 (anti-Sca1 biotin) and then enriched using Anti-Biotin MicroBeads by autoMACS Pro (Miltenyi Biotec). The enriched cells, stained with antibodies, were sorted by BD FACSAria III.

| Transplantation
All recipients (CD45.1 + , C57BL/6 background) were lethally irradiated (9 Gy, RS2000, Rad Source) at least 4 h, but less than 24 h before transplantation. MPP (400 cells/mouse) from Rosa LSL-Hoxb5-EGFP/+ mouse or Mx1cre/Rosa LSL-Hoxb5-EGFP/+ mouse for primary transplantation and donor-derived CD48 + CD11b + SK cells (2000 cells/mouse) from the primary recipients for secondary transplantation were retroorbitally transplanted into the recipients with the enriched Sca1 À helper cells (0.25 million/mouse, CD45.1 + ). For third transplantation, total BM cells (10 million/mouse) from the secondary recipients were retro-orbitally transplanted into the third recipients. To induce Hoxb5 expression, the primary recipients were intraperitoneally injected with polyinosinic-polycytidylic acid (pIpC) (250 μg/mouse) every other day for six times starting from the 5th day before transplantation, until the 5th day after transplantation. Recipients were fed with the water added with trimethoprim-sulfamethoxazole for 1 month after irradiation. reported. 16,17 Gene set enrichment analysis (GSEA) and gene-ontology (GO)-enrichment analysis (clusterProfiler package) were performed as described. 18,19 Spearman correlation coefficient between population was used for correlation analysis. Correlation analysis was performed by cor() function of R. 3.2 | Hoxb5 results in the occurrence of a de novo CD11b + CD48 + SK cell type associated with the long-term engraftable feature

|
To investigate the cellular mechanism, we analysed the blood progenitor cells in the primary recipients at Week 20 post-transplantation. We discovered a de novo donor-derived Sca1 + c-kit + population cells, which simultaneously expressed CD11b and CD48 surface markers. Certainly, this cell type is not identified in natural blood cells in the absence of Hoxb5 expression ( More importantly, the donor-derived CD11b + CD48 + SK cells can still be detected in the BM of the secondary recipients ( Figure 2E).
These results indicate that the de novo CD11b + CD48 + SK cell type is engraftable in the secondary recipients.

| DISCUSSION
In this study, we explored the role of Hoxb5 in the MPP cell context.  14 which also showed no T lineage-biased feature. Furthermore, there was also no T lineage-biased differentiation in CD19cre/Rosa LSL-Hoxb5-EGFP/+ mouse, though T lymphocytes were abundantly produced in the recipients which were transplanted with pro-pre-B cells sorted from CD19cre/Rosa LSL-Hoxb5-EGFP/+ mouse.
Hence, these results suggested that Hoxb5 empowers different cell types with different cell fates.
Reportedly, ectopic expression of either Sox17 or miR-125a in MPP can confer a self-renewal ability but eventually resulted in haematological malignancies. Sox17 eventually led to leukemogenesis within 374 days after transplantation and MiR-125a-induced MPN displayed a complex manner of oncogene dependency. 7,36 Interestingly, no haematological malignancies were found in the recipients transplanted with the Hoxb5-expressing MPP. Thus, the self-renewal feature activated by Hoxb5 might be insulated from oncogenesis.
We also tested the engraftment potential of HOXB5-expressing human MPP in immunodeficient animals. Unfortunately, the HOXB5expressing human MPP failed to recapitulate the long-term engraftment phenotype of Hoxb5-expressing murine MPP (data not shown). One possible reason is that the function of HOXB5 is not conservative between human and mouse species. However, we cannot exclude another possibility that HOXB5-overexpressing human MPP need a humanized bone marrow micro-environment for HOXB5-reprgramming, which is not available in current immunodeficient animal models.
In conclusion, our study reveals a rare role of Hoxb5 in empowering self-renewal capacity on MPP, which provides insights into converting blood progenitors into alternative engraftable cell sources.