Constance Jean Eaves
Relevant Degree Programs
Great Supervisor Week Mentions
Today, for #TeacherAppreciationDay, I thank my supervisor Connie Eaves @EavesLab for not only educating me about stem cells & critical scientific research but for empowering me as a woman leader ready to face the world. @UBCGradSchool @ScienceVancity
#GreatSupervisor Week @UBC: I want to acknowledge the humility & creativity with which my supervisor Connie Eaves @EavesLab leads students.
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
Intra-tumoural biological, transcriptional and genomic heterogeneity are hallmarks of human breast cancers. However, tumour propagating activity appears confined to subsets of cells within each tumour. This finding is thought to indicate a persistence of mechanisms that maintain a hierarchical growth and differentiation structure in the normal mammary gland. Because little is known about the responses of either primary normal or malignant human mammary cells to existing therapies, this thesis sought to examine the intrinsic sensitivity of different purified human mammary colony-forming cell (CFC) types and tumours derived from them to ionizing radiation.Luminal progenitor (LP) CFCs were found to be ~1.5-fold more radioresistant than basal cell (BC) CFCs and LPs also showed evidence of checkpoint adaptation, slower repair activity and greater predisposition of γH2AX foci accumulation. Two human breast cancer cell lines (MDA-MB231 and SUM149) and a non-tumorigenic human mammary cell line (MCF-10A) were all found to be more radioresistant than the normal LP-CFCs. CFCs isolated from 8-week tumours generated in mice transplanted with normal human BCs or LPs transduced with KRASG¹²D showed even greater radioresistance and this was further increased in serially passaged derivative lines with more aggressive growth properties. To examine the responsiveness of malignant cells with tumor-initiating cell (TIC) activity in vivo, a dose-response analysis was first undertaken of their frequencies in the MDA-MB231 and SUM149 cells. Limiting dilution analysis (LDA) and single-cell transplants showed the frequency of TICs in both to be very high (
The normal mammary gland contains “stem cells” with extensive in vivo growth and bi-lineage differentiation potential and a surface phenotype of basal cells (BCs). BCs also contain cells with more limited growth and differentiation activity in vitro. An analogous luminal-restricted progenitor (LPs) subset has surface characteristics of both basal and luminal cells. I hypothesized that the growth and differentiation activity displayed by individual mammary epithelial cells from both subsets would be highly diverse, and that the properties of tumours produced from these cells would be affected by their cell of origin. To address this hypothesis, I first developed a lentiviral-mediated barcoding strategy that involves transducing each cell with a unique 27-base pair non-coding DNA sequence so that the number of its clonal progeny can be inferred from high-throughput sequencing data obtained on the progeny of bulk-transduced populations. The use of “spiked-in” control cells carrying a known barcode provided an internal calibration for clone size calculations and allowed clones of ≥100 cells to be reliably detected. Application of this strategy to normal mouse and human mammary cells identified expected bi-lineage clones but an unanticipated predominance of lineage-restricted clones produced in primary transplants. These experiments also revealed that many clones apparent in secondary hosts were not detected in the primary hosts, indicating their origin from cells with very delayed growth activity. Application of the barcoding strategy to normal human BCs and LPs transduced with lentiviruses encoding KRASG¹²D ± PI3KCAH¹⁰⁴⁷R ± TP53R²⁷³C showed tumour formation in subsequently transplanted immunodeficient mice was rapid (within 8 weeks) and efficient from both cell types (8-12/18 donors, 1/200-1/4,000 transduced cells). However, tumours generated from LPs contained larger clones than tumours generated from BCs. Surprisingly, none of the LP-derived tumours were ERα⁺ (typical of luminal-like breast cancers) whereas 60% of the BC-derived tumours were. Earlier analysis of xenografts of similarly transduced cells revealed changes in both the number and phenotype of the cells present. Taken together, these findings underscore the diverse regenerative activity of normal mammary cells and provide definitive evidence that the cell of origin can affect the properties of human breast tumours generated using identical oncogenes.
Transplantation of hematopoietic cells is a critical component of treatments used to cure a range of malignancies and congenital disorders. Cells with rapid but short-term repopulating ability (STRCs) are the main source of neutrophils and platelets early post-transplant, but methods to increase their availability are lacking. In this study the use of NOD/SCID-IL-2Receptor-γchain-null mice engineered to produce human interleukin-3, granulocyte-macrophage colony-stimulating factor and Steel factor as hosts of CD34⁺ cells was shown to improve the quantification of human STRCs. This mouse was found to support a 5-fold higher human myeloid cell output at early time-points post-transplant, sufficient to assess total engraftment from an analysis of circulating human cells. This strategy was then used to determine the optimal protocol for mobilizing STRCs in normal adult human donors, comparing administration of granulocyte colony stimulating factor (G-CSF) plus plerixafor (P) to administration of P alone. Blood and marrow samples were obtained from 10 normal adult donors before, during, and after treatment and then evaluated for their content of CD34⁺ cells, colony-forming cells (CFCs), long-term culture-initiating cell activity, and cells with in vivo STRC activity. The results show all activities were maximally increased in the blood 4 hours after administration of P, with or without pre-treatment with G-CSF. In vivo assessment showed that administration of P led to a 30-fold increase in STRC activity over baseline levels, with further enhancement (90-fold over baseline) by prior G-CSF treatment. The ability of currently available growth-factor (GF)-containing HSC expansion protocols to support the expansion of STRCs was then assessed. The results revealed a significant (10-fold) loss of STRC activity after being cultured for 7 to 10 days, in spite of an extensive increase in CD34⁺ cells and CFCs. This GF-induced loss of in vivo STRC activity occurred within 24 hours, and was paralleled in time and amount by a loss of CFC homing to the bone marrow. Together these findings provide a further understanding of STRC biology and provide a foundation for developing improved yields of manipulated STRCs for clinical use.
The concept of stem cell self-renewal was developed from clonal tracking of hematopoietic stem cell (HSC) divisions in vivo 50 years ago. However, protocols to expand these cells in vitro without loss of their stem cell properties have remained elusive. A number of factors contribute to this inability. Key among these is a lack of knowledge of the critical molecular characteristics that distinguish HSCs from hematopoietic progenitors as well as how the control of the fundamental biological programs of survival, division and differentiation are integrated in HSCs. Using a combination of single-cell tracking, transcriptomics, and in vivo readouts applied to highly enriched mouse HSCs, we now show that their survival, proliferation, and maintenance of stem cell properties are mechanistically dissociable. Discovery of a protocol that allows input numbers of functionally intact human HSC numbers to be maintained for 3 weeks in vitro using defined growth factors, was then leveraged to design single human HSC cell tracking and functional analyses. The results of these showed that for human HSC, as in the mouse model, survival, proliferation, and maintenance of stem cell status are mechanistically dissociable, and controlled in a combinatorial manner. We then developed a panel of mass cytometry detectors to enable >40 surface and intracellular proteins to be simultaneously measured at single cell resolution. Using this panel, we identified some of the signaling intermediates activated by growth factors that differentially control human HSC biological responses assessed in high-throughput assays. Correlation of the molecular properties, surface phenotypes and functional activities of CD34+ subsets have further revealed a surprising degree both of heterogeneity within each phenotype and overlap between phenotypes. In some cases, the results suggest a given phenotype contains distinct subsets and a broader scheme of differentiation pathways than suggested by current models of human hematopoietic cell differentiation. Finally, we identify CD33+ as a novel marker which demarcates the most potent human HSC within the current best phenotypic enrichment strategy. These results lay a foundation on which future HSC expansion strategies can be constructed, and have implications for the development of leukemia.
Mouse hematopoietic stem cells (HSCs) undergo a post-natal transition in several properties, including a marked reduction in their self-renewal activity. To investigate the molecular basis of this difference, we devised a single strategy to isolate fetal and adult HSCs at similarly high frequencies. This strategy, involving fluorescence-activated cell sorting of cells with a CD45⁺EPCR⁺CD48-CD150⁺ (ESLAM) phenotype, allows isolation of HSCs at a frequency of ~1 in 2 from all developmental time points tested (mouse embryonic day (E) 14.5 to adult). Comparison of differentially expressed genes in primitive populations of fetal and adult hematopoietic cells showed that heightened expression of Hmga2 was a feature of fetal as compared to adult HSCs. We also identified let-7 microRNAs (miRNAs) and a negative regulator of their biogenesis, Lin28b, to be expressed in an opposite and similar pattern to Hmga2, respectively. Since Hmga2 is a well-established target of let-7 miRNAs, we hypothesized that the Lin28b-let-7-Hmga2 axis plays a central role in the determination of fetal versus adult HSC self-renewal identity. We also found that Lin28 overexpression in adult HSCs restores a higher, fetal-like, self-renewal potential in them, and this effect is phenocopied by direct overexpression of Hmga2. Conversely, HSCs from fetal Hmga2-/- mice display a prematurely acquired adult-like self-renewal activity. Importantly, we show that Lin28-mediated activation of Hmga2 expression, which is responsible for the activation of a fetal-like self-renewal potential in adult HSCs, is not the mechanism by which Lin28 reprograms adult HSCs to undergo fetal-like B-cell differentiation. Together, these findings suggest a model of development in which Lin28b acts as a master regulator and Hmga2 serves as a more specific downstream modulator of HSC self-renewal. These findings may help inform strategies to improve the therapeutic use of HSCs. Furthermore, since Lin28b and Hmga2 are oncogenes, we speculate that the fetal/neonatal specific pattern of expression of these genes may contribute to the pathogenesis of pediatric leukemias.
No abstract available.
No abstract available.
High-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Recently, it has been demonstrated that engineered NUP98-HOXA10hd expression stimulates >1,000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin⁻Sca-1⁺c-kit⁺ cells. In this thesis I coupled such ability of engineered NUP98-HOXA10hd expression, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. I discovered that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at near unit efficiency in cultures initiated with single cells. The clonally expanded HSCs showed preservation of normal proliferation kinetics in vitro and consistent balanced contributions long-term to the lymphoid and myeloid lineages in vivo without evidence of leukemogenic transformation. Preservation of a normal proliferating HSC phenotype allowed their re-isolation in large numbers at 25% purity. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process. Although there is growing excitement about the prospect of in vitro expansion of HSCs and their use to enhance the safety and application of transplant-based therapies, deleterious consequences of such manipulations remain unknown. Thus, I further examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. I observed ~10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tert⁻/⁻ HSCs 6 months before. The second generation leukocytes also showed elevated expression of γH2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type or Tert⁻/⁻ HSCs, suggesting that HSC replication post-transplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity upon prolonged self-renewal stimulation.
No abstract available.
No abstract available.
Prospective Student Info Sessions
Faculty of Medicine Information SessionDate: Tuesday, 08 December 2020
Time: 11:00 to 12:00
UBC’s Faculty of Medicine is a global leader in both the science and the practice of medicine, and is home to more than 1,700 graduate students across over 20 graduate programs. In this session hosted by Dr Michael Hunt, Associate Dean, Graduate and Postdoctoral Education, we’ll provide an overview of the diverse array of graduate programs available, including cutting-edge research experiences in the biosciences, globally recognized population health education, quality health professional training, as well as certificate and online training options. Dr Hunt will also be joined by program advisors from across the faculty to take an inside look at the application process and provide some application tips to help make your application as strong as possible.