Final Report Summary - BM 3D-AGING (Multidimensional Analysis of Bone Marrow Microenvironmental Dynamics during Aging)
The principal scientific objectives of the project could be summarized as follows:
1. To characterize the structural changes in the stromal microenvironment of the bone marrow (BM) during aging through the use of novel imaging approaches
2. To analyze potential functional defects in vascular and mesenchymal stromal cell populations of the bone marrow as a consequence of aging
3. To visualize hematopoietic stem cells in their BM niches in aged mice
4. To transfer the unique imaging techniques previously developed by the fellow to the host institution
In addition, a fundamental aim of the project was to facilitate the establishment and reintegration of the fellow (César Nombela-Arrieta, CNA) in the European research community through the obtention of a position as Senior Investigator in an educational or scientific institution. Collectively the majority of the aims of the grant have been achieved.
In the initial phases of the project the fellow managed to transfer the main imaging techniques developed during his postdoctoral period in Harvard Medical School, Boston. This was the primary step towards the completion of the work as these techniques were absolutely indispensable for the proposed work, and not available in the host institution. The experimental protocols and technologies required for three-dimensional imaging of extensive regions of bone marrow (BM) tissues have not only been implemented but also further optimized and developed. We implemented procedures to enable complete tissue clearing of our samples thereby enhancing light penetration and imaging quality. Most importantly, through a fruitful collaboration with Computer Vision lab and Scientific Center for Optical and Electron Microscopy (SCOPE-M) of the Swiss Federal Technical School (ETH) Zurich we have developed state of the art tools for the fast, quantitative and unbiased computational analyses of large-scale 3D image datasets. Altogether, these technologies have allowed us to characterize the composition and topology of the bone marrow stromal compartment in homeostasis with unprecedented detail. The results of this basic investigation are summarized in a manuscript currently under revision in Nature Communications entitled 3D quantitative spatial analysis of hematopoiesis-supporting stromal cells in the BM microenvironment. The novel findings reported here have fundamental implications on the current understanding of the physiological relevance and mode of action of BM stromal cells in the regulation of hematopoiesis.
Furthermore, by employing these methods we have further analyzed the alterations that arise in the BM stromal framework during aging. Our observations suggests that, contrary to previous reports, mesenchymal stromal cells do not change in number and the integrity of their cellular networks is preserved. Nonetheless, major modifications in the aged BM vascular compartment were detected. We are currently exploring the potential implications of the observed expansion of transitional vessels in the described deregulation of hematopoietic homeostasis in the BM during aging.
Our studies are revealing that beyond being structurally altered, functional modifications arise in BM stromal cells, and specifically in their production of hematopoietic-supportive factors as a consequence of aging. We recently performed a large-scale transcriptomic analysis of the four principal subtypes of BM stromal cells, namely arterial endothelial cells (AECs), sinusoidal endothelial cells (SECs), mesenchymal CXCL12 abundant reticular cells (CARc) and so called “PαS” mesenchymal progenitor cells, isolated from young, adult and aged mice, using next generation sequencing techniques. The preliminary analyses of these experiments allow us to conclude that: i) aging results in progressive and significant changes in the production of hematopoietic supportive factors and extracellular matrix proteins by all stromal cells ii) during aging CARc and SECs increase expression of inflammatory related genes, which may lead to critical changes in the BM milieu and modifications of stem cell properties iii) aging-related alterations in the transcriptomic signatures of stromal cells partially resemble those observed during viral and bacterial infections. Collectively these results are consistent with the model of “inflammaging”, which posits that ageing-related perturbations of hematopoiesis are hypothesized to derive from a basal pro-inflammatory milieu.
Finally, the funding provided by the Marie Curie Career Integration Grant was absolutely pivotal in allowing the fellow to establish an independent research program. During the course of the funding period CNA has been appointed Assistant Professor in Hematology of the Medical Faculty of the University and University Hospital Zurich (since November 2015). Moreover, he has secured over 1 million CHF in competitive third-party research funding from several funding agencies including Swiss National Foundation, Swiss Cancer League or Novartis Foundation for Biomedical Research. Finally, he currently runs a laboratory formed by four graduate students and has directed one master thesis.
1. To characterize the structural changes in the stromal microenvironment of the bone marrow (BM) during aging through the use of novel imaging approaches
2. To analyze potential functional defects in vascular and mesenchymal stromal cell populations of the bone marrow as a consequence of aging
3. To visualize hematopoietic stem cells in their BM niches in aged mice
4. To transfer the unique imaging techniques previously developed by the fellow to the host institution
In addition, a fundamental aim of the project was to facilitate the establishment and reintegration of the fellow (César Nombela-Arrieta, CNA) in the European research community through the obtention of a position as Senior Investigator in an educational or scientific institution. Collectively the majority of the aims of the grant have been achieved.
In the initial phases of the project the fellow managed to transfer the main imaging techniques developed during his postdoctoral period in Harvard Medical School, Boston. This was the primary step towards the completion of the work as these techniques were absolutely indispensable for the proposed work, and not available in the host institution. The experimental protocols and technologies required for three-dimensional imaging of extensive regions of bone marrow (BM) tissues have not only been implemented but also further optimized and developed. We implemented procedures to enable complete tissue clearing of our samples thereby enhancing light penetration and imaging quality. Most importantly, through a fruitful collaboration with Computer Vision lab and Scientific Center for Optical and Electron Microscopy (SCOPE-M) of the Swiss Federal Technical School (ETH) Zurich we have developed state of the art tools for the fast, quantitative and unbiased computational analyses of large-scale 3D image datasets. Altogether, these technologies have allowed us to characterize the composition and topology of the bone marrow stromal compartment in homeostasis with unprecedented detail. The results of this basic investigation are summarized in a manuscript currently under revision in Nature Communications entitled 3D quantitative spatial analysis of hematopoiesis-supporting stromal cells in the BM microenvironment. The novel findings reported here have fundamental implications on the current understanding of the physiological relevance and mode of action of BM stromal cells in the regulation of hematopoiesis.
Furthermore, by employing these methods we have further analyzed the alterations that arise in the BM stromal framework during aging. Our observations suggests that, contrary to previous reports, mesenchymal stromal cells do not change in number and the integrity of their cellular networks is preserved. Nonetheless, major modifications in the aged BM vascular compartment were detected. We are currently exploring the potential implications of the observed expansion of transitional vessels in the described deregulation of hematopoietic homeostasis in the BM during aging.
Our studies are revealing that beyond being structurally altered, functional modifications arise in BM stromal cells, and specifically in their production of hematopoietic-supportive factors as a consequence of aging. We recently performed a large-scale transcriptomic analysis of the four principal subtypes of BM stromal cells, namely arterial endothelial cells (AECs), sinusoidal endothelial cells (SECs), mesenchymal CXCL12 abundant reticular cells (CARc) and so called “PαS” mesenchymal progenitor cells, isolated from young, adult and aged mice, using next generation sequencing techniques. The preliminary analyses of these experiments allow us to conclude that: i) aging results in progressive and significant changes in the production of hematopoietic supportive factors and extracellular matrix proteins by all stromal cells ii) during aging CARc and SECs increase expression of inflammatory related genes, which may lead to critical changes in the BM milieu and modifications of stem cell properties iii) aging-related alterations in the transcriptomic signatures of stromal cells partially resemble those observed during viral and bacterial infections. Collectively these results are consistent with the model of “inflammaging”, which posits that ageing-related perturbations of hematopoiesis are hypothesized to derive from a basal pro-inflammatory milieu.
Finally, the funding provided by the Marie Curie Career Integration Grant was absolutely pivotal in allowing the fellow to establish an independent research program. During the course of the funding period CNA has been appointed Assistant Professor in Hematology of the Medical Faculty of the University and University Hospital Zurich (since November 2015). Moreover, he has secured over 1 million CHF in competitive third-party research funding from several funding agencies including Swiss National Foundation, Swiss Cancer League or Novartis Foundation for Biomedical Research. Finally, he currently runs a laboratory formed by four graduate students and has directed one master thesis.