Novel Strategies for Identification and Isolation of Murine Adult HSCs and Embryonic Pre-HSCs
- Author(s): Karimzadeh, Alborz
- Advisor(s): Inlay, Matthew A
- et al.
Billions of blood cells are produced in the body on a daily basis. At the top of the hematopoietic hierarchy are the hematopoietic stem cells (HSCs) that are able to self-renew, give rise to all downstream progenitors, and engraft long-term upon transplantation into a conditioned recipient. HSC transplantation (HSCT) can be employed to repopulate a defective blood system with a healthy one. In fact, HSCT has the potential to treat any disease that is inherent to the blood system. Yet due to the complications associated with it, HSCT is often utilized as a last-resort option. In recent years, the advent of induced pluripotent stem cells (iPSCs) has promised a definitive solution to HSCT-related complications by introducing the prospect of patient-specific iPSC-derived HSCs (iHSCs). Encouraging advancements have produced iHSCs that exhibit HSC-like properties, however, generation of iHSCs with therapeutic potential has failed due to a lack in robust BM engraftment potential.
Generated iHSCs share similar characteristics to the embryonic precursors to HSCs, pre-HSCs, which also lack BM engraftability. Understanding the natural pathways that drive maturation of pre-HSCs into BM-engraftable HSCs can shed much-needed light on the necessary signals required for the generation of therapeutic iHSCs. However, our understanding of the development of the hematopoietic system is incomplete. The ability to better isolate HSCs and pre-HSCs would allow improved characterization of these cells and highlight the molecular differences between them, and perhaps, identify the defining factors in pre-HSC maturation.
Here we report the use of CD11a as a potent marker of adult HSCs and embryonic pre-HSCs. We introduce an efficient strategy for FACS-purification of adult HSCs. This new strategy addresses the issues associated with HSC sorting in conditions where conventional HSC markers fail. Furthermore, we provide evidence for the efficacy of CD11a in combination with another HSC marker, endothelial protein C receptor (EPCR; also known as CD201), for the isolation of all BM HSCs without the need for additional HSC markers.
CD11a can also be used to identify embryonic pre-HSCs. Here we demonstrate that CD11a greatly improves the purity of pre-HSCs in a rare sorted population. Moreover, our sorting strategy with CD11a allows the isolation of all pre-HSCs from all the tissues and the timepoints that are relevant to pre-HSC biology. Furthermore, we confirm the identity of our putative pre-HSC population with the use of an entirely in vivo model (instead of a commonly-used ex vivo assay which may not reveal pre-HSC-specific activity). Based on our findings, we propose that improved characterization of cellular and molecular players in the developing liver niche can benefit our understanding of pre-HSC maturation. Altogether, we establish CD11a as an indispensable marker for adult HSC and embryonic pre-HSC identification and isolation.