Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) and generate blood cells for the entire lifespan of an animal. HSCs are mostly quiescent, but can self-renew and generate all lineages of the hematopoietic system. Their clinical significance lies in their potential to engraft after transplantation and reconstitute the blood and immune system in patients with hematological malignancies, immune deficiencies or hemoglobin abnormalities. Despite significant progress in our understanding of mechanisms involved in self-renewal, differentiation and quiescence, a coherent picture of how these mechanisms act in concert to regulate steady-state function and homeostatic responses of HSCs has not emerged yet. Importantly, reliable renewal or even maintenance of HSCs in vitro remains challenging.
The identification of dozens of cytokines and of more than 200 genes affecting HSC function in knockout studies, as well as multiple publications on genome-wide expression and epigenetic signatures, still leaves significant gaps in our understanding. From a clinical-translational perspective, it is essential to bridge these gaps in our knowledge to devise strategies to maintain HSCs in vitro. This would have enormous implications for the current practice of allogeneic and autologous bone marrow transplantation, as well as gene therapy and genome editing targeting HSCs. Our lab has previously shown that culture in the presence of reduced calcium concentrations allowed striking maintenance of HSC function over at least two weeks. Furthermore, calcium controlled expression of the master hematopoietic tumor suppressor, TET2, while TET2 expression affected the response of HSCs to extracellular calcium. Despite this progress, quantitative expansion of functional HSCs was not achieved through low-calcium culture, suggesting other barriers to self-renewal exist in vitro. The goal of this thesis is to gain a deeper understanding in the barriers to self-renewal of HSCs, both in vitro and in vivo.
During fetal life, HSC develop in the fetal liver (FL), where they expand, and home to the BM around birth. As FL HSCs exhibit more self-renewal than adult HSCs, we examined the response of these cells to calcium and to deletion of Tet2 in hopes of identifying barriers to self-renewal in the adult. Surprisingly, we observed that FL HSCs have very distinct calcium physiology compared to adult HSCs and could not be maintained in vitro in any calcium concentration. Only in the presence of low-calcium and after deletion of Tet2 could maintenance of functional FL HSCs be achieved in vitro. This is in sharp contrast to adult HSCs, which were maintained in low-calcium conditions, and in which deletion of Tet2 attenuated maintenance in these conditions. These data indicate more profound differences in the biology of fetal versus adult HSCs than previously appreciated, and suggest that recapitulating the extensive renewal capacity of FL HSCs in adult HSCs may not possible with identical culture conditions.
Further studies into mechanisms involved in HSC maintenance in low-calcium conditions revealed that these conditions attenuated the propensity of HSCs to differentiate into megakaryocytes (Mk), hyperploid cells that generate platelets essential to hemostasis. Whereas most hematopoietic lineages arise through successive, increasingly lineage-committed progenitors, Mks can derive rapidly and directly from HSCs. Direct megakaryopoiesis from HSCs occurs in particular in response to inflammatory stimuli, such as interferon signaling. We therefore tested the hypothesis that direct Mk specification is a barrier to HSC self-renewal that is alleviated at least in part by culture in low-calcium conditions.
Interferon signaling has been reported to induce direct megakaryopoiesis and also rapidly recruits HSCs into cell cycle. HSCs are also known to be susceptible to replication stress and ensuing DNA damage. We therefore examined the connection between DNA damage responses (DDR) and direct megakaryopoiesis. We discovered that interferon signaling induced DNA damage through replication stress in vivo, whereas irradiation rapidly induced Mk commitment in HSCs. These findings established a connection between a DDR and direct megakaryopoiesis. Furthermore, quiescent HSCs are subject to a physiological DDR caused by hypertranscription, while in vitro culture induced replication stress. Inflicting additional DNA damage in HSCs in vitro or in vivo rapidly induced expression of Mk markers. Even in the absence of additional DNA damage, pharmacological blockade of the G2 phase of the cell cycle induced MK differentiation and hyperploidy in HSCs, but apoptosis in progenitors. Part of the underlying mechanisms are post-transcriptional. Increased protein expression of the Mk lineage transcription factor GATA1 was induced by both DNA damage and G2 arrest, and preceded upregulation of Gata1 mRNA and other Mk genes. Expression of GATA1 protein is at least in part mediated by the integrated stress response (ISR), which modulates translation. Together these findings show that direct megakaryopoiesis from HSCs can be stimulated by DNA damage-induced G2 arrest and is at least partially post-transcriptionally regulated.
As our findings suggested that direct megakaryopoiesis, among others induced by a DDR, limits HSC maintenance, we initiated studies to identify the mechanism underlying the DDR in cycling HSCs. We discovered that cycling HSCs are particularly prone to misincorporation of uracil into DNA in vivo and in vitro. Supplementation with thymidine in vitro decreased uracil incorporation, attenuated the DDR, and strikingly increased the maintenance of multipotential HSCs in vitro. Thymidine supplementation also lowered expression of CD41, a marker of Mk-committed HSCs. These data establish a profound role of a uracil-induced DDR in HSCs and indicate that direct commitment to the Mk lineage is inversely correlated with functional HSC maintenance. The DDR, however, was not affected by low-calcium conditions, indicating other pathways in addition to DDR signaling can likely lead to direct Mk specification from HSCs.
Collectively, our work establishes that preventing direct Mk commitment in HSCs, either by preventing uracil incorporation or by culture in low-calcium conditions, enhances HSC maintenance, thereby establishing that the propensity to directly engage the Mk pathway is a barrier to HSC maintenance.
These findings will have important implications for future efforts at manipulating HSCs in vitro and at in vivo hematopoietic recovery after insults such as irradiation, chemotherapy, and inflammation. Furthermore, two arguments support the notion that this work may have uncovered an important tumor suppressor mechanism. First, the folate cycle, which provides thymidine and prevents uracil misincorporation, is upregulated in most cancers and targeted by several drugs, while folate deficiency is not oncogenic. This suggests that limiting the supply of thymidine in HSCs prevents inadvertent expansion and malignant transformation. Second, our findings indicate that DNA-damaged HSCs, in part through uracil misincorporation, rapidly generate a lineage essential to immediate organismal survival, thus removing potentially mutated cells from the HSC pool to avoid malignant transformation.
Finally, we also attempted to study the in vivo relevance of calcium regulation of HSCs. HSCs reside in the BM, and as bone is the main calcium buffering in the body. We therefore initiated studies to investigate whether changes in bone turnover, potentially mediated by changes in microenvironmental calcium concentration, affect HSCs function. Although difficult to directly correlated with calcium conditions in vitro, our findings indicate that both increased and decreased bone turnover do affect HSC function in vivo. Interestingly, bone turnover differentially affects HSCs with mutation in Tet2. These observations may have clinical significance as recent studies revealed that premature menopause, which is associated with increased bone turnover, accelerates the development of clonal hematopoiesis, a condition caused among others by mutation in Tet2.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/fr77-g374 |
| Date | January 2023 |
| Creators | Garyn, Corey Michael |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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