Clonal Level Lineage Commitment of Mouse Hematopoietic Stem Cells in Vivo

Abstract 27

Hematopoietic stem cells (HSCs) sustain the blood and immune systems through a complex differentiation process. This process involves several steps of lineage commitment and forms a paradigm for understanding cellular development, differentiation, and malignancy. While this step-wise differentiation has been extensively studied at the population level, little is known about the lineage commitment of individual HSC clones. The importance of understanding HSC differentiation at the clonal level has been raised by several recent studies suggesting that individual HSCs differentially contribute to various blood cell types and that the aggregate HSC differentiation at the population level is an amalgamation of the diverse lineage commitments of individual HSC clones. The distinct differentiation of individual HSCs may also be accentuated by their regulatory microenvironments, HSC niche. HSC niche may not affect all HSCs in an organism equally, and may instead act directly on resident HSC clones through direct contact or by tuning local cytokine concentrations. Knowledge of HSC clonal level lineage commitment will reveal new insights into HSC regulatory mechanisms and will improve our understanding of aging, immune deficiency, and many hematopoietic disorders involving an unbalanced hematopoietic system. Here, we provide a comprehensive map of in vivo HSC clonal development in mice.

The clonal map was derived from the simultaneous tracking of hundreds of individual mouse HSCs in vivo using genetic barcodes. These unique barcodes were delivered into HSCs using a lentiviral vector to obtain a one-to-one mapping between barcodes and HSCs. Barcoded HSCs were then transplanted into recipient mice using standard procedures. Genetic barcodes from donor derived HSCs and their progenies were examined twenty-two weeks after transplantation using high-throughput sequencing.

We found that the dominant differentiation of HSC clones is always present in pre-conditioned mice. In these recipients, a small fraction of engrafted HSCs become dominantly abundant at the intermediate progenitor stages, but not at the HSC stage. Thus, clonal dominance is a characteristic of HSC differentiation but not of HSC self-renewal. Additionally, the dominant differentiation of HSC clones exhibits distinct expansion patterns through various stages of hematopoiesis. We provide evidence that observed HSC lineage bias arises from dominant differentiation at distinct lineage commitment steps. In particular, myeloid bias arises from dominant differentiation at the first lineage commitment step from HSC to MPP, whereas lymphoid bias arises from dominant differentiation at the last lineage commitment step from CLP to B cells.

We also show that dominant differentiation and lineage bias are interrelated and together delineate discrete HSC lineage commitment pathways. These pathways describe how individual HSC clones produce differential blood quantities and cell types. Multiple clonal differentiation pathways can coexist simultaneously in a single organism, and mutually compensate to sustain overall blood production. Thus, the distinct HSC differentiation characteristics uncovered by clonal analysis are not evident at the population level. We have also identified the lineage commitment profiles of HSC clones belonging to each pathway. These profiles elucidate the cellular proliferation and development of HSCs at the clonal level and demonstrate that distinct modes of HSC regulation exist in vivo.

In summary, our in vivo clonal mapping reveals discrete clonal level HSC lineage commitment pathways. We have identified the cellular origins of clonal dominance and lineage bias, which may be the key hematopoietic stages where blood production and balance can be manipulated. These discoveries based on clonal level analysis are unexpected and unobtainable from conventional studies at the population level. Together, they open new avenues of research for studying hematopoiesis.