Tracking dynamic interactions between haematopoietic stem cells and osteoblasts: molecular regulation of stem cell position and fate

Most blood cells are short lived and are constantly replaced by the balanced production of new progeny arising from blood stem cells through several commitment and differentiation steps. Blood cells are known to be quiescent and proliferate infrequently relative to their progeny. When they do so, they give rise to more stem cells (self-renewal) or to differentiating progeny. Blood stem cells finely tune the balance between quiescence, self-renewal and differentiation by sensing tissue damage and adjusting progeny generation to physiological and stress-induced demand. In the same way that a comfortable home contributes to family's happiness and the work environment influences employees' performance, correct positioning of blood stem cells in specific niches within the bone marrow space is crucial to ensure their proper functioning. Several studies indicate that bone-making cells, called osteoblasts, have an important role in regulating blood stem cell numbers and function; however, the few studies aimed at pinpointing blood stem cell location within the bone marrow have reported a wide range of positions and even when most observed cells are in the vicinity of osteoblasts only few are clearly in direct contact. As a number of stem cell niches observed in invertebrate model organisms or in different mammalian tissues function through direct interaction between the stem and niche cells, a controversy has arisen whether osteoblasts truly are the main component of the blood stem cell niche, provided such a niche exists at all. I have recently developed an in vivo imaging methodology that allows me to visualise transplanted blood stem cells within the bone marrow space immediately after their injection into the blood, and I can 'watch' the behaviour of the same cells until few days later. I have collected evidence that switching on or off certain molecular signals within transplanted blood stem cells can modulate their function by means of affecting their positioning. The current proposal seeks to apply and further develop in vivo imaging of blood stem cells in order to gain a definite answer on the existence, location and function of the blood stem cell niche. We will use as our working model transgenic mice expressing over-physiological levels of the Wnt inhibitor Dkk1 in all their osteoblasts, which have been shown to have decreased blood stem cell function and specifically self-renewal ability. We know that transplanted blood stem cells localize abnormally in Dkk bone marrow, and we plan to test whether this altered positioning is the cause of their malfunction. We will perform detailed time-lapse tracking of transplanted stem cells to ask whether cells closer to osteoblasts are more quiescent (less likely to divide) compared to those further away, and we will take advantage of novel photoconversion techniques to tag stem cells and their progeny residing in different locations and subsequently compare their function. Moreover, as current in vivo imaging technologies do not allow sub-cellular resolution, we will investigate whether co-culture of blood stem cells and osteoblasts from normal or Dkk mice can be used as a surrogate system to study in greater detail at least some of the events observed in the mice, and we will use them to closely track blood stem cells behaviour and morphology under normal and disadvantageous conditions. We will perform genome wide expression analysis of stroma and stem cells from self-renewal permissive or repressive cultures or mice, and we will focus on genes known to regulate cell-cell and cell-environment interactions. As a result, we will gain a comprehensive picture of the relationship between osteoblasts and blood stem cells, we will investigate whether stem cell position directly affects function, and we will learn what molecules are involved in the process.

Even though the presence of a niche for haematopoietic stem cells (HSC) was postulated nearly four decades ago and several studies present evidence for the role of osteoblasts as HSC regulators, final proof that these cells are the main component of the HSC niche is still lacking. Indeed, a growing number of voices now speculate that there may be no such a thing as a single, spatially defined bone marrow HSC niche. By crossing the col2.3GFP osteoblast reporter mice with the col2.3 Dkk1 (Dkk) strain we have the ability to observe HSC residing in a non-supportive environment. Our in vivo imaging of HSC transplanted into Dkk recipients revealed an abnormal localization pattern, thus we can now investigate whether there is a direct relationship between HSC position relative to osteoblasts and function. We will test the reconstitution ability of HSC residing in wild type vs. Dkk bone marrow and HSC transplanted into osteoblast reporter mice will be tracked over time in order to study whether HSC residing near osteoblasts maintain a higher degree of quiescence. We will perform photoconversion experiments to selectively label HSC near to or far from osteoblasts, harvest them and test their function to determine whether position in the peri-osteoblastic space confers specific functional capabilities. We will establish whether co-cultures of HSC with wild type and Dkk osteoblasts are a good surrogate for the phenotypes observed in vivo and we will use them to track sub-cellular resolution events that correspond with the loss of HSC self-renewal potential. We will perform expression profiling analysis of self-renewal permissive or repressive osteoblasts and corresponding HSC in order to gain a comprehensive picture of the molecules involved in the cross-talk between the two cell types and we will test whether overexpression or repression of cell adhesion genes can determine HSC positioning and function.

Academic impact is described in the section 'Academic beneficiaries'. Health impact: Translational medical research/patients The knowledge acquired with the proposed study will lead to a better understanding of the mechanisms regulating HSC fate and function through their interaction with bone marrow osteoblasts. Information about in vivo regulation of HSC function will allow generation of better in vitro systems for HSC culture and expansion, which are today lacking. Such cultures would have a tremendous impact fro all patients requiring regenerative medicine treatments such as bone marrow transplantation. A higher availability of transplantable HSC would reduce waiting times and increase the number of treated patients, especially of those carrying rare HLA haplotypes. Moving forward The work presented in this proposal can be performed independently by my group, based on my current expertise and knowledge. However, it will largely benefit from day-to-day interactions with other members of the Division of Cell and Molecular Biology (in particular Dr. Hugh Brady, who is expert in haematopoiesis) and of the College (in particular Dr. Luis Pizarro, in the Centre for Integrative Systems Biology, located in my same building and Prof. Paul French, heading the Photonics group the Department of Physics in the same campus). In vivo imaging of HSC within the bone marrow microenvironment is still a newly developed technique and is usually welcomed with high enthusiasm. I already established numerous local and international collaborations (thanks to the fact that bone marrow cells can travel overnight without significantly suffer) from my previous position in Boston and I am looking forward to continuing such work within the UK scientific community. On a higher level of collaborative work, the proposed study will provide basic knowledge about HSC regulation, which will be immediately available for development into translational research. Dissemination All obtained results will be submitted for publication in peer-reviewed journals through open access whenever possible. Stem cells and in vivo imaging networks are being developed at the national (e.g. London Stem Cell Network, London Regenerative Medicine Network, UK Stem Cell Network, BioimagingUK) and international level (e.g. EuroBioimaging, EuroSystem, ISSCR) and the proposed work will fit well within the interests of both and will be presented at relevant conferences. Moreover, I intend to continue my previous involvement in outreach activities and I will be happy to participate to similar activities organised by Imperial College. In particular, the presented work will be amenable to presentation to the public during discussions on the social and ethical implications of stem cell research. Professional skills The researcher working on this project will gain state-of-the-art training in stem cell research, confocal and two photon microscopy, gene expression profiling, image and data analysis. Moreover, he/she will be immersed into the diverse and active scientific community of the South Kensington campus, which offers a choice of several high quality seminars on the most diverse scientific topics and, through information circulated by members of the local stem cell networks, will have access to seminars presented by visiting highly-regarded international stem cell researchers. Such experience should contribute to the development of all skills needed to develop his/her future career path.