Mitochondrial dynamics in hematopoietic stem cells Mariana Justino Lage de Almeida Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2019 © 2019 Mariana Justino Lage de Almeida All rights reserved ABSTRACT Mitochondrial dynamics in hematopoietic stem cells Mariana Justino Lage de Almeida Hematopoietic stem cells (HSCs) take on the extraordinary role of sustaining life-long production of blood cells. Despite their indisputable therapeutic potential, HSC biology is poorly understood, and the field remains limited by the inability to maintain, expand, or generate HSCs in vitro. The aim of this study was to elucidate a particular gap in our understanding of the organellar cell biology of HSCs, specifically the role and function of the mitochondria. Several signaling pathways and biological processes converge onto the mitochondria, yet these organelles were found to be largely dispensable in HSCs on the basis of their predominantly glycolytic metabolism and reports of low mitochondrial content. Our studies show that MitoTracker Green (MTG), a frequently used fluorescent dye to measure mitochondrial mass in hematopoietic populations, is effluxed by HSCs resulting in their systematic and deceptive enrichment in the subset of cells with the lowest MTG fluorescence. Using dye-independent methods we discovered that HSCs have elevated mitochondrial content despite their reliance on glycolysis for ATP production. Moreover, mechanisms of mitochondrial quality control and clearance by autophagy appear to be comparatively lower in HSCs than in any other hematopoietic population we analyzed, suggesting HSCs maintain their mitochondria over time. To investigate the function of mitochondria in HSCs we generated mice with disruption of mitofusins (MFN) 1 and 2. These proteins are key mediators of mitochondrial fusion, a process that in coordination with mitochondrial fission regulates mitochondrial size, number, and function. Mice with deletion of Mfn1 and Mfn2 (DKO) die perinatally, are pale in appearance and their HSCs show complete loss of regenerative capacity. Several processes linked to dysfunctional mitochondrial fusion and known to be tightly regulated in HSCs are altered in these mutants, including mitochondrial morphology, mitochondrial mass, proliferation, and altered metabolism. Interestingly, one allele of Mfn1 is sufficient to rescue the hematopoietic function and lethality of DKO mice, while one allele of Mfn2 only rescues myeloid reconstitution. Taken together, our findings highlight the importance and complexity of mitochondrial function and dynamics in HSCs and have contributed to the recently increased appreciation of a vital role for mitochondria in HSCs. Table of Contents List of Figures ................................................................................................................ iv List of Tables ............................................................................................................... viii List of Acronyms and Abbreviations ........................................................................... ix Acknowledgments ....................................................................................................... xiv 1. Introduction: Principles of hematopoiesis and hematopoietic stem cells ........ 1 2. Introduction: Mitochondria and cellular health ................................................. 27 3. Mitochondrial content in HSCs ........................................................................... 49 i 4. Mitochondrial degradation by autophagy in HSCs ........................................... 79 5. Deletion of Mitofusin 1 and 2 in the hematopoietic system ........................... 109 6. Conclusions........................................................................................................ 169 7. Materials and Methods ...................................................................................... 172 ii References .................................................................................................................. 188 Appendix. List of differentially expressed genes in 3W and/or DKO FL HSC ....... 221 iii List of Figures Chapter 1. Principles of hematopoiesis and hematopoietic stem cells Figure 1. Classical model of hematopoiesis……………….…………………………...……23 Chapter 3. Mitochondrial content in HSCs Figure 1. MTG fluorescence in hematopoietic populations…………………………...……56 Figure 2. MTG fluorescence of HSCs in the presence of verapamil…………………….…58 Figure 3. Effect of verapamil in MTG retention………………………………………………59 Figure 4. HSC activity in vivo based on MTG fluorescence………………………………...60 Figure 5. Validation of qPCR assay for determination of mtDNA:nDNA ratio……………61 Figure 6. Relative mtDNA:nDNA in MTGHi and MTGLo fractions ……………………...…..62 Figure 7. Relative mtDNA:nDNA in HSCs and more differentiated populations………….63 Figure 8. Relative mtDNA:nDNA in Map17+HSCs…………………………………………..63 Figure 9. Mitochondrial nucleoids in hematopoietic populations………………………….64 Figure 10. Validation of MitoDendra2 mice as a reporter of mitochondrial content………65 Figure 11. MitoDendra2 fluorescence in hematopoietic populations………..…………….66 Figure 12. HSC activity in vivo based on MitoDendra2 fluorescence……………………..67 Figure 13. MTG fluorescence in human CB hematopoietic populations………………….68 Figure 14. Relative mtDNA:nDNA in human and mouse hematopoietic cells in early developmental stages………………………………………………………………………....69 Figure 15. Metabolic profile of adult BM HSCs………………………………………………71 iv Chapter 4. Mitochondrial degradation by autophagy Figure 1. Mitochondrial mass in young and old mice………………………………….……..85 Figure 2. Effect of autophagy inhibition on mitochondrial mass………………………….…86 Figure 3. Mitochondrial autophagy in vivo…………………………………………………….87 Figure 4. HSC activity in mt-Keima BM fractions………….…………………………….……89 Figure 5. Induction of mitophagy……………………………………………………...……….90 Figure 6. Effect of mitophagy induction……………………………………………….………91 Figure 7. Characterization of Parkin-deficient mice………………………………...………..93 Figure 8. Frequency and number of HSCs in BM of Parkin-deficient mice………………..94 Figure 9. HSC activity of Parkin-deficient BM in competitive transplantation assays……94 Figure 10. Mitochondrial content in Parkin-deficient mice…………………………………..95 Figure 11. Effect of Park2 deletion on cell proliferation………………………………………96 Figure 12. Effect of Park2 deletion in mitochondrial morphology…………………………...96 Figure 13. Mitochondrial turnover in CD150High and CD150Low HSCs……………………...98 Figure 14. Mitochondrial turnover in Mfn2-/-hematopoietic populations……………………99 Chapter 5. Deletion of Mitofusin 1 and 2 in the hematopoietic system Figure 1. Generation of mitofusins-deficient mice………………………………………….116 Figure 2. Hematopoietic deletion of Mfn1 and Mfn2 in mice…………………………….…117 Figure 3. Embryo appearance and erythroid differentiation……………………………….118 Figure 4. Mitochondrial content in late erythropoiesis…………………………...…………120 Figure 5. Myeloid progenitors in the FL……………………………………………………...121 Figure 6. HSC frequency in the FL……………………………………………………...……122 v Figure 7. Expression of intracellular Ki-67 in 3W and DKO…………………………..……123 Figure 8. Colony forming capacity of 3W and DKO HSCs……………………...………….124 Figure 9. Donor chimerism in primary competitive transplant……………………………..125 Figure 10. Donor lineage reconstitution in primary competitive transplant……….………126 Figure 11. Analysis of lymphocytes in competitive transplantation……………………….128 Figure 12. Donor chimerism in recipient hematopoietic tissues…………………………..129 Figure 13. Lymphoid progenitors in 3W FL…………………………………...……………..130 Figure 14. T cell development in 3W FL……………………………………………………..131 Figure 15. 3W donor HSC frequency in the BM of recipient mice…………………………132 Figure 16. Expression of myeloid-bias HSC markers in 3W HSCs………………………..133 Figure 17. 3W donor HSC frequency in the liver and spleen of recipient mice…………...135 Figure 18. Donor chimerism and lineage reconstitution in secondary transplant……..…136 Figure 19. 3W chimerism in tertiary transplants………………………………………….…137 Figure 20. Inducible deletion of Mfn1 and Mfn2 in mice………………………...………….138 Figure 21. Survival and excision post Poly(I:C) injection…………………………………..139 Figure 22. Analysis of adult DKO hematopoietic tissues…………………………..………140 Figure 23. Excision of Mfn1 and Mfn2 in Mx1-Cre donor BM…………………………..….141 Figure 24. HSC frequency and numbers in the BM of Mx1-Cre donor mice……….……..142 Figure 25. Deletion of mitofusins in adult BM after transplant……………..………………143 Figure 26. Mitochondrial morphology and content in FL HSCs……………………………145 Figure 27. Intracellular calcium concentration in FL LSKs…………………………………147 Figure 28. Metabolic profile of FL Lineage- cells……………………………………………149 Figure 29. Mitochondrial membrane potential……………………………………………....150 vi Figure 30. Sample variation in 3W and DKO HSCs…………………………………….…..151 Figure 31. Differently expressed genes in 3W and DKO FL HSCs………………….…….152 Figure 32. Upregulated pathways in 3W and DKO HSCs……………………………….…153 Figure 33. DKO-specific pathway analysis……………………………………………...…..154 Figure 34. Heatmap of differently expressed genes in 3W and DKO HSCs………….…..155 Figure 35. Heatmap of differently expressed genes uniquely in DKO HSCs…………….156 Chapter 6. Conclusions Figure 1. Graphical summary I……………………………………………………………….169 Figure 2. Graphical summary II………………………………………………………………170 vii List of Tables Table 1. Oligonucleotides used in