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Human Stem Cell Models Identify Targets of Healthy HUMAN STEM CELL MODELS IDENTIFY TARGETS OF HEALTHY AND MALIGNANT HEMATOPOIETIC REGULATION BY JENNIFER C REID, BSc A Thesis Submitted to the School of Graduate Studies In Partial Fulfillment of the Requirements For the Degree Doctor of Philosophy McMaster University ÓCopyright by Jennifer C Reid, April 2020 JC Reid <- PhD Thesis ❖ McMaster University $ Biochemistry Descriptive Note McMaster University BACHELOR OF SCIENCE (2015) Hamilton, Ontario (Biomedical Research Specialization, Co-op) Title: Use of human stem cell models to identify targets of healthy and malignant hematopoietic regulation Author: Jennifer C. Reid Supervisor: Dr. Mickie Bhatia Number of pages: xii, 163 ii JC Reid <- PhD Thesis ❖ McMaster University $ Biochemistry AbstrAct Hematopoiesis is the highly regenerative process of producing billions of blood cells each day, including white blood cells, red blood cells, and platelets. Given the relatively short life span of these mature cells, hematopoiesis is dependent on stem and progenitor cells to generate renewed progeny, which represents a tightly regulated process. This includes cell intrinsic and external factors, and where dysregulation can lead to anemia and cancer. As such, the hematopoietic hierarchy has been intensely studied for nearly a century and represents a gold standard model of cell fate and developmental biology, in research and clinical applications. Cellular models, such as in vitro culture and human-mouse xenografts in vivo, have been developed to explain complex phenomena pertaining to hematopoiesis and also interrogate processes which are too invasive to study in humans. Hematopoietic generation is required beyond sustaining homeostasis, and progenitors can be damaged through cytotoxic injuries such as radiation and standard chemotherapy, and also undergo leukemic transformation. There are two main treatment modalities for leukemia patients (a) receiving a stem cell transplant, and (b) drug or radiation-based therapy. In the former, shortages of donors and stem cells has remained an unmet clinical need for decades. In the latter, selective targeting of genetic mutations has become a successful standard-of-care in leukemias such as chronic myelogenous leukemia and acute promyelocytic leukemia. However, in the most common adult hematologic malignancy, chronic lymphocytic leukemia (CLL), similar targeting therapies have not been developed. Altogether, shortages of stem cells from healthy donors, chemotherapy-induced immune dysfunction, and a lack of targeted therapies, all reinforce the immediate need for innovative cellular models to address these clinical problems. To generate additional sources of human hematopoietic progenitors for laboratory study, human PSCs have been used. Unlike hematopoietic progenitor cells collected from healthy and leukemic donors, human pluripotent stem cells (PSC) can be easily propagated and expanded in vitro. PSCs can generate hematopoietic progenitor cells, but they remain poorly understood and have not been robustly applied to solve the aforementioned deficiencies related to patient treatment. Importantly, the biological regulation of both hematopoiesis and PSCs has been experimentally confirmed to significantly deviate between humans and other animals, such as mice, further reinforcing the importance of human- specific cell models of hematopoiesis. Therefore, I hypothesized that human stem cell models provide a focused approach to interrogate the regulation of hematopoiesis from the apex of the hierarchy, which can be used to understand the promotion of healthy hematopoiesis and understand malignant transformation. We began by providing evidence that a discovery platform based on human PSCs with a specific leukemic driver mutation, trisomy 12 (tri12), enabled unprecedented elucidation of cell autonomous pathways for precision oncology, specifically in the context of tri12 CLL. Together with traditional gene expression analysis, we also developed machine learning algorithms to molecularly dissect pathways specific to tri12 in PSC and CLL, that could only be found using human isogenically-controlled PSC with a genetic leukemic driver in vitro. Two actionable targets were therapeutically applied to target tri12 CLL patient samples and tri12 PSCs both in vitro and upon xenotransplantation as tumors into immunocompetent mice. iii JC Reid <- PhD Thesis ❖ McMaster University $ Biochemistry We then investigated what is necessary to improve PSC-derived hematopoietic progenitors to extend their application beyond in vitro models, and also to address the clinical shortage of transplantable stem cells. We discovered that PSC-derived progenitors fail to survive even 24 hours following bone marrow transplantation, while they can survive and proliferate for weeks in vitro. We then determined PSC-derived progenitors do not have a key receptor that healthy progenitors have, called CXCR4. By correcting CXCR4 signaling, we observed enhanced PSC-derived progenitor function and survival in the bone marrow of human-mouse xenografts and a transcriptional shift toward human progenitor profiles. Our results reveal CXCR4 is an actionable target for transplantable progenitors with in vivo function. To investigate hematopoietic dysfunction, which is a pervasive side-effect of radiation and chemotherapy in vivo, we developed a third human cellular model using long-term human-mouse xenografts given these broad effects cannot be recapitulated in vitro. We sampled xenografts at multiple time points before and during the recovery process after treatments and observed drastic yet reproducible changes in the composition of human progenitors and also mature human hematopoietic cells, which reflected clinical leukemia patient experiences. Furthermore, using single cell transcriptional and protein analysis, we were able to identify unique subsets of human progenitors which may prove to be the key hematopoietic regenerating cells upon further biological investigation of these actionable targets. Validation of clinically relevant interventions which aid in the process of healthy hematopoietic regeneration in vivo are the next step in this work. Collectively, the data presented within this thesis offer a deeper conceptualization of human stem cell models and the deconvolution of several complex components of hematopoietic regulation. This work has revealed novel, clinically relevant, and actionable targets to ultimately enable the promotion of healthy hematopoiesis on multiple fronts. iv JC Reid <- PhD Thesis ❖ McMaster University $ Biochemistry Acknowledgements Mick, I want to thank you for all the once-in-a-lifetime memories; from litterbox and oompa loompas to creative brain storming and motivational speeches. I have learned so much from you, and I am grateful for that. You have taught me so much, and also let me get messy and learn from my mistakes. I appreciate that a thesis should capture the 'hard skills' and data generated, but I would be amiss not to expressly thank-you for the even longer list of 'soft skills' and personal development that are not directly mentioned in this thesis. I’d also like to thank my committee members, Drs. Thomas Hawke and Greg Steinberg, who have asked out-of-the-box questions and advised me during my graduate studies. I have admired your character since my undergrad days, and I am so grateful to have such positive roles models. My graduate success would not have been possible without my lab family. Diana and Allison, you have become true friends and I am so grateful for everything we’ve shared. I want to thank Jong-Hee and Kyle for taking a chance on me and trusting me to articulate and publish their work. I want to thank everyone whom I have had the pleasure to work with, including current members and alumni of the Bhatia lab; JB, Borko, Macky, Luca, Deanna, Gena, Yeonjoon, Juan, Andrew, Lili, Mio, and the bhatiabhaddies; you make my day. To all the brave patients, and their families, who participated in clinical trials and donated valuable tissue samples used in this and other studies, I am truly grateful. Justin, whether you’re cooking me dinner or at the gym while I’m at the lab, I wouldn’t have been able to finish this degree without your love and support. Here’s my chance to publicly apologize for every time I was late leaving the lab. Last, but not least, I want to thank my family, who continuously promote me to become a better version of myself. ❖ “There is no point in starting something without ambition. ... Ambition never goes away. It may shuffle off, grumbling, feet dragging, only to slide across into something else – usually the next project. It doesn’t take ‘no’ for an answer. ... One last word to all you nascent writers out there. Ambition is not a dirty word. Piss on compromise. Go for the throat. Write with balls, write with eggs. Sure, it’s a harder journey but take it from me, it’s well worth it. – Steven Erickson [Steve Rune Lundin], 2007 ❖ v JC Reid <- PhD Thesis ❖ McMaster University $ Biochemistry Table of Contents Descriptive Note ........................................................................................................................ ii Abstract ........................................................................................................................................ iii Acknowledgements ................................................................................................................... v Table of Contents .....................................................................................................................
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