The goals of this proposal are to (1) develop new methods to study and genetically perturb functional bone marrow hematopoietic (blood-forming) stem cells (HSCs) in a dish, and (2) support the completion of my scientific training and transition to research independence at the interface of stem cell biology and hematology. My long- term objective is to develop new approaches for hematological disease treatment. To achieve this, we need to define the biological mechanisms responsible for the unique ability of HSCs to regenerate the entire blood system and understand how genetic corruption of these mechanisms result in hematological diseases. Besides the pathogenic consequences of HSC dysfunction, understanding how healthy HSCs function is also clinically important in order to improve the safety and efficacy of bone marrow or HSC transplantation, a curative therapy for various blood diseases including bone marrow failures, anemias, and leukemias. This research project will build on a novel synthetic culture system that I have recently developed, which affords long-term and large-scale expansion of transplantable mouse HSCs (functionally assayed by their ability to engraft and reconstitute the hematopoietic system within a bone marrow conditioned [irradiated] recipient).
In Aim 1, I will develop new approaches to prospectively isolate transplantable HSCs from this long-term culture and characterize the cellular heterogeneity within the system.
In Aim 2, I will optimize targeted gene editing in transplantable mouse HSCs by combining HSC expansion culture with recent CRISPR/Cas9 gene editing technologies to assess how gene editing influences HSC function in vivo following transplantation.
In Aim 3, I will apply these new tools to develop novel in vitro and in vivo disease modeling systems to study the pre- malignant hematological state known as Clonal Hematopoiesis. This will be achieved by combining ex vivo HSC expansion and targeted gene mutation with HSC transplantation into non-conditioned recipients, which will allow me to investigate how clinically-relevant genetic mutations alter HSC activity within the native (non-irradiated) bone marrow and decipher the autonomous and non-autonomous mechanisms underlying this hematological phenotype. Through the development and study of these new biological models, I aim to identify new therapeutic strategies to prevent, treat, and cure hematological diseases. My mentored training K99-phase will be performed at the world-class Institute for Stem Cell Biology and Regenerative Medicine at Stanford University School of Medicine under the guidance of Professor Hiromitsu Nakauchi, a world-renowned HSC expert. Besides new technical training, the K99-phase offers the opportunity for me to expand my academic and management skills. These will facilitate my successful transition to a R00- phase independent research position. This award will thereby enable my long-term objective of using experimental hematology and stem cell research to better understand the biological mechanisms regulating hematopoiesis in order to identify new strategies to improve treatment options for hematological diseases.
As a long-lived self-renewing stem cell population that sustains blood system homeostasis, bone marrow hematopoietic stem cells (HSCs) are not only therapeutically important as the functional unit in bone marrow transplantation, but are also clinical relevant as the cell-of-origin for various hematological diseases that are caused by a gradual accumulation of somatic mutations. However, until my recent discovery of a novel HSC expansion culture system, long-term growth of functional HSCs outside the body was not possible, which has been a major barrier to further investigation of this important cell population. This research project will develop my novel HSC expansion culture system into a multifaceted toolkit to study HSCs ?in a dish?, and apply this system to evaluate how genetic changes within HSCs influence functional activity and hematological disease progression, with the aim of identifying new strategies to treat and prevent blood disorders.
