Stable Long-Term Blood Formation by Stem Cells in Murine Steady-State Hematopoiesis

Abstract

Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady‐state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady‐state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification‐mediated‐polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV‐injected animals for up to 24 months, indicating stable marking of murine steady‐state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long‐term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short‐lived progenitor cells. Myelosuppressive treatment of mice prior to LV‐injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady‐state hematopoiesis. Stem Cells2012;30:1961–1970

Clonal dynamics of native haematopoiesis

“…Stochastically, a fraction of these clones can get recruited for blood production, where they undergo commitment and a massive proliferative burst to produce detectable PB progeny. Our findings of successive and polyclonal long-term behaviour are supported by irradiation marking experiments 27,28 and by more recent studies involving in vivo lentiviral tagging 29 . Similarly, variance analyses have predicted that haematopoiesis is maintained by a large number of haematopoietic clones 30,31 .…”

Long-term in vivo monitoring of mouse and human hematopoietic stem cell engraftment with a human positron emission tomography reporter gene

“…A competitive mouse bone marrow transplantation (BMT) study was chosen to test whether hdCK3mut can detect transplanted cells during early hematopoietic reconstitution (24)(25)(26)(27)(28). Donor cells were generated by treating mice with 5-flourouracil 5 d preharvest for HSC enrichment.…”

“…We examined whether reporter cells in BMT-recipient mice retained the PET reporter function through serial imaging in vivo. Consecutive scans and peripheral blood analysis were obtained at 4, 15, 27, and 32 wk post-BMT allowing for detection of both short-and long-lived reporter HSCs (27,28).…”

Altered Erythro-Myeloid Progenitor Cells Are Highly Expanded in Intensively Regenerating Hematopoiesis

“…However, several experimental findings have indicated a more complex organization of the immature hematopoietic cells and also challenged the idea that the extensive self-renewal capacity is a unique property of HSCs (Adolfsson et al, 2005;England et al, 2011;Yamamoto et al, 2013;Kim et al, 2015). It was also demonstrated that the undisturbed murine hematopoiesis is maintained by multiple clones acting in parallel (Zavidij et al, 2012;Sun et al, 2014) without any significant contribution from HSCs. Busch et al (2015) also demonstrated that undisturbed adult hematopoiesis is largely sustained by cells downstream of HSCs, and Schoedel et al (2016) reported a long-term hematopoiesis occurring in the absence of HSCs while, in contrast, Sawai et al (2016) and Akinduro et al (2018) presented the data supporting the continuous contribution of HSCs for steady state hematopoiesis.…”