2019 Fiscal Year Annual Research Report
Cross-organ HSC trafficking during definitive hematopoiesis formation
| Project/Area Number |
18J20690
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| Research Institution | Kumamoto University |
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Principal Investigator |
瀬崎 真衣子 熊本大学, 医学教育部, 特別研究員(DC1)
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| Project Period (FY) |
2018-04-25 – 2021-03-31
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| Keywords | HSC trafficking / definitive hematopoiesis / 3D whole-organ imaging / lineage tracing / mathematical modeling |
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| Outline of Annual Research Achievements |
This project utilizes forefront technology (3D organ-wide imaging, deep learning-based quantification analysis, mathematical modeling/simulation) to clarify various aspects of hematopoietic stem cell (HSC) trafficking that have so far remained elusive.
Colonization of potential HSCs on the outside of the femur bone was discovered earliest by E16.5, and we suspect these to be the initial “colonizers” arriving from the fetal liver. A protocol devised for their separation from the HSCs already within the bone marrow (BM) proved successful, and both populations when subjected to single-cell RNA-seq and PCA identified clusters of HSCs that are distinct from each other phenotypically (HSC surface markers) and functionally (lineage bias and reconstitution potential).
Furthermore, our imaging data of major hemogenic organs using a HSPC tracer crossed with other stromal mouse lines offers by far the most descriptive documentation of HSC-niche cell relations reported during ontogeny. There are certain aspects of neonatal HSC character that may directly relate to adult-type HSCs (i.e., regulatory mechanisms and niche relations). Our imaging and functional (i.e., transplantation) data allow novel inferences to be made regarding the initial stages of definitive hematopoiesis formation, especially in relation to the heterogeneous nature of HSCs and their hierarchical organization.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The past two years have been devoted to collecting descriptive data, including fluorescent 3D images of major hemogenic organs and flow cytometry analysis of phenotypic HSCs tracked through fetal to neonatal development. Using the above mentioned data we additionally sought to formulate a mathematical model to simulate HSC trafficking. Currently, we are at the final stages of devising this model.
Single-cell RNA sequencing performed last year provided us with a list of differentially expressed genes for each HSC cluster, which we have partially validated (protein expression). Much of our efforts this year have been directed towards functional studies including single-cell colony formation assays (in vitro) and transplantation of our cells of interest into lethally irradiated mice and analysis of their lineage output (in vivo). Particularly, the latter is “the method” to test for functionality and reconstitution potential, and several rounds have been completed. The next question pertains to their biological significance and whether there are any differences in their contribution to the adult hematopoietic system. We are currently preparing materials, including a new mouse line to trace their lineage and optimizing experimental conditions for this.
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| Strategy for Future Research Activity |
The ultimate question lies in the nature of these HSC subtypes distinct on several levels (i.e., protein expression of HSC markers, differentially expressed genes, functional experiments) that have been identified in the BM of a P2 mouse femur. Their ontogeny, role and contribution towards the adult hematopoietic system are key aspects to be explored, now that their phenotypical identification is nearing completion. For this, a highly sensitive lineage tracing system is required. We plan to use the confetti mouse crossed with an inducible novel HSPC tracer. Upon injection of tamoxifen, potential HSPCs and their progenitors will be labeled randomly with one of four colors (CFP, GFP, YFP, RFP). The ratio of each color will allow speculation into their ontogeny. A preliminary demo of this system has been attempted in the adult. These mice are being bred and optimization of tamoxifen concentrations (for embryos) are being determined. A barcode system with even greater sensitivity is in parallel being devised and its use under contemplation.
Additionally, quantification of all imaging data will be conducted on site at the University of Zurich under the supervision of Dr. Cesar Nombela-Arrieta starting October 2020. The collaboration is supported by the Challenge Overseas Program granted by JSPS. Furthermore, simulation of HSC trafficking via a mathematical model will be wrapped up and experimentally validated. To examine HSC-niche cell associations, stromal cell dynamics (i.e., vasculature, lepR+ cells) may also be included as an additional variable.
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