Evolving as a Scientist and a Mentor.

Connie Eaves sets high standards for herself, her science, and her colleagues, which has fueled a stellar career that counts as successes insights into basic stem cell biology, important discoveries in leukemia and breast cancer, and a cohort of trainees that she considers family. Cell editor Lara Szewczak caught up with Connie, the recipient of the 2019 Canada Gairdner Wightman Award, to discuss her dual passions for stem cell biology and mentoring talented young scientists. Annotated excerpts from this conversation are presented below.

Stem Cells, Genome Editing, and the Path to Translational Medicine.

The derivation of human embryonic stem cells (hESCs) and the stunning discovery that somatic cells can be reprogrammed into human induced pluripotent stem cells (hiPSCs) holds the promise to revolutionize biomedical research and regenerative medicine. In this Review, we focus on disorders of the central nervous system and explore how advances in human pluripotent stem cells (hPSCs) coincide with evolutions in genome engineering and genomic technologies to provide realistic opportunities to tackle some of the most devastating complex disorders.

Timeline: iPSCs--The First Decade.

Research into induced pluripotent stem cells (iPSCs) has expanded at a remarkable pace in the decade since Shinya Yamanaka and Kazutoshi Takahashi first reported their groundbreaking discovery in 2006. This Timeline highlights the key events in the development of this field, including basic insights into the production of iPSCs and how they have been applied to improve our understanding and treatment of human disease.

Onconephrology: The intersections between the kidney and cancer.

Onconephrology is a new subspecialty of nephrology that recognizes the important intersections of kidney disease with cancer. This intersection takes many forms and includes drug-induced nephrotoxicity, electrolyte disorders, paraneoplastic glomerulonephritis, and the interactions of chronic kidney disease with cancer. Data clearly demonstrate that, when patients with cancer develop acute or chronic kidney disease, outcomes are inferior, and the promise of curative therapeutic regimens is lessened. This highlights the imperative for collaborative care between oncologists and nephrologists in recognizing and treating kidney disease in patients with cancer. In response to this need, specific training programs in onconephrology as well as dedicated onconephrology clinics have appeared. This comprehensive review covers many of the critical topics in onconephrology, with a focus on acute kidney injury, chronic kidney disease, drug-induced nephrotoxicity, kidney disease in stem cell transplantation, and electrolyte disorders in patients with cancer.

Pluripotent stem cells progressing to the clinic.

Basic experimental stem cell research has opened up the possibility of many diverse clinical applications; however, translation to clinical trials has been restricted to only a few diseases. To broaden this clinical scope, pluripotent stem cell derivatives provide a uniquely scalable source of functional differentiated cells that can potentially repair damaged or diseased tissues to treat a wide spectrum of diseases and injuries. However, gathering sound data on their distribution, longevity, function and mechanisms of action in host tissues is imperative to realizing their clinical benefit. The large-scale availability of treatments involving pluripotent stem cells remains some years away, because of the long and demanding regulatory pathway that is needed to ensure their safety.

Pluripotent stem cells in disease modelling and drug discovery.

Experimental modelling of human disorders enables the definition of the cellular and molecular mechanisms underlying diseases and the development of therapies for treating them. The availability of human pluripotent stem cells (PSCs), which are capable of self-renewal and have the potential to differentiate into virtually any cell type, can now help to overcome the limitations of animal models for certain disorders. The ability to model human diseases using cultured PSCs has revolutionized the ways in which we study monogenic, complex and epigenetic disorders, as well as early- and late-onset diseases. Several strategies are used to generate such disease models using either embryonic stem cells (ES cells) or patient-specific induced PSCs (iPSCs), creating new possibilities for the establishment of models and their use in drug screening.

A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species.

Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3ß inhibitors, two calpain inhibitors, and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin, and the GSK3ß inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.

An autoimmune stem-like CD8 T cell population drives type 1 diabetes.

CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained, and the molecular programmes defining the autoimmune T cell state, are unknown. In type 1 diabetes, ß-cell-specific CD8 T cells destroy insulin-producing ß-cells. Here we followed the fate of ß-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN), which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas, where they differentiate further and destroy ß-cells. Whereas transplantation of as few as 20 autoimmune progenitors induced type 1 diabetes, as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived, and stem-like autoimmune progenitors must continuously seed the pancreas to sustain ß-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes.

Reprogramming cellular identity for regenerative medicine.

Although development leads unidirectionally toward more restricted cell fates, recent work in cellular reprogramming has proven that one cellular identity can strikingly convert into another, promising countless applications in biomedical research and paving the way for modeling diseases with patient-derived stem cells. To date, there has been little discussion of which disease models are likely to be most informative. Here, we review evidence demonstrating that, because environmental influences and epigenetic signatures are largely erased during reprogramming, patient-specific models of diseases with strong genetic bases and high penetrance are likely to prove most informative in the near term. We also discuss the implications of the new reprogramming paradigm in biomedicine and outline how reprogramming of cell identities is enhancing our understanding of cell differentiation and prospects for cellular therapies and in vivo regeneration.

Identification of stem cell populations in sweat glands and ducts reveals roles in homeostasis and wound repair.

Sweat glands are abundant in the body and essential for thermoregulation. Like mammary glands, they originate from epidermal progenitors. However, they display few signs of cellular turnover, and whether they have stem cells and tissue-regenerative capacity remains largely unexplored. Using lineage tracing, we here identify in sweat ducts multipotent progenitors that transition to unipotency after developing the sweat gland. In characterizing four adult stem cell populations of glandular skin, we show that they display distinct regenerative capabilities and remain unipotent when healing epidermal, myoepithelial-specific, and lumenal-specific injuries. We devise purification schemes and isolate and transcriptionally profile progenitors. Exploiting molecular differences between sweat and mammary glands, we show that only some progenitors regain multipotency to produce de novo ductal and glandular structures, but that these can retain their identity even within certain foreign microenvironments. Our findings provide insight into glandular stem cells and a framework for the further study of sweat gland biology.

Spermatogonial stem cell self-renewal and development.

Spermatogenesis originates from spermatogonial stem cells (SSCs). Development of the spermatogonial transplantation technique in 1994 provided the first functional assay to characterize SSCs. In 2000, glial cell line-derived neurotrophic factor was identified as a SSC self-renewal factor. This discovery not only provided a clue to understand SSC self-renewing mechanisms but also made it possible to derive germline stem (GS) cell cultures in 2003. In vitro culture of GS cells demonstrated their potential pluripotency and their utility in germline modification. However, in vivo SSC analyses have challenged the traditional concept of SSC self-renewal and have revealed their relationship with the microenvironment. An improved understanding of SSC self-renewal through functional assays promises to uncover fundamental principles of stem cell biology and will enable us to use these cells for applications in animal transgenesis and medicine.

Base-Edited CAR7 T Cells for Relapsed T-Cell Acute Lymphoblastic Leukemia.

BACKGROUND: Cytidine deamination that is guided by clustered regularly interspaced short palindromic repeats (CRISPR) can mediate a highly precise conversion of one nucleotide into another - specifically, cytosine to thymine - without generating breaks in DNA. Thus, genes can be base-edited and rendered inactive without inducing translocations and other chromosomal aberrations. The use of this technique in patients with relapsed childhood T-cell leukemia is being investigated. METHODS: We used base editing to generate universal, off-the-shelf chimeric antigen receptor (CAR) T cells. Healthy volunteer donor T cells were transduced with the use of a lentivirus to express a CAR with specificity for CD7 (CAR7), a protein that is expressed in T-cell acute lymphoblastic leukemia (ALL). We then used base editing to inactivate three genes encoding CD52 and CD7 receptors and the ß chain of the αß T-cell receptor to evade lymphodepleting serotherapy, CAR7 T-cell fratricide, and graft-versus-host disease, respectively. We investigated the safety of these edited cells in three children with relapsed leukemia. RESULTS: The first patient, a 13-year-old girl who had relapsed T-cell ALL after allogeneic stem-cell transplantation, had molecular remission within 28 days after infusion of a single dose of base-edited CAR7 (BE-CAR7). She then received a reduced-intensity (nonmyeloablative) allogeneic stem-cell transplant from her original donor, with successful immunologic reconstitution and ongoing leukemic remission. BE-CAR7 cells from the same bank showed potent activity in two other patients, and although fatal fungal complications developed in one patient, the other patient underwent allogeneic stem-cell transplantation while in remission. Serious adverse events included cytokine release syndrome, multilineage cytopenia, and opportunistic infections. CONCLUSIONS: The interim results of this phase 1 study support further investigation of base-edited T cells for patients with relapsed leukemia and indicate the anticipated risks of immunotherapy-related complications. (Funded by the Medical Research Council and others; ISRCTN number, ISRCTN15323014.).

Regulatory T cells suppress myeloma-specific immunity during autologous stem cell mobilization and transplantation.

ABSTRACT: Autologous stem cell transplantation (ASCT) is the standard of care consolidation therapy for eligible patients with myeloma but most patients eventually progress, an event associated with features of immune escape. Novel approaches to enhance antimyeloma immunity after ASCT represent a major unmet need. Here, we demonstrate that patient-mobilized stem cell grafts contain high numbers of effector CD8 T cells and immunosuppressive regulatory T cells (Tregs). We showed that bone marrow (BM)-residing T cells are efficiently mobilized during stem cell mobilization (SCM) and hypothesized that mobilized and highly suppressive BM-derived Tregs might limit antimyeloma immunity during SCM. Thus, we performed ASCT in a preclinical myeloma model with or without stringent Treg depletion during SCM. Treg depletion generated SCM grafts containing polyfunctional CD8 T effector memory cells, which dramatically enhanced myeloma control after ASCT. Thus, we explored clinically tractable translational approaches to mimic this scenario. Antibody-based approaches resulted in only partial Treg depletion and were inadequate to recapitulate this effect. In contrast, a synthetic interleukin-2 (IL-2)/IL-15 mimetic that stimulates the IL-2 receptor on CD8 T cells without binding to the high-affinity IL-2Ra used by Tregs efficiently expanded polyfunctional CD8 T cells in mobilized grafts and protected recipients from myeloma progression after ASCT. We confirmed that Treg depletion during stem cell mobilization can mitigate constraints on tumor immunity and result in profound myeloma control after ASCT. Direct and selective cytokine signaling of CD8 T cells can recapitulate this effect and represent a clinically testable strategy to improve responses after ASCT.

A prospective multi-institutional study of eculizumab to treat high-risk stem cell transplantation-associated TMA.

ABSTRACT: High-risk, complement mediated, untreated transplant-associated thrombotic microangiopathy (hrTMA) has dismal outcomes due to multi-organ dysfunction syndrome (MODS). The complement C5 blocker eculizumab shows promising results in hrTMA, but has not been prospectively studied in hematopoietic stem cell transplant (HCT) recipients. We performed the first multi-institutional prospective study in children and young adults to evaluate eculizumab as an early targeted intervention for hrTMA/MODS. We hypothesized that eculizumab would more than double survival in HCT recipients with hrTMA, compared to our prior study of prospectively screened, untreated hrTMAs serving as historical controls. HrTMA features (elevated terminal complement (sC5b-9) and proteinuria measured by random urine protein/creatinine ratio (≥1mg/mg)) were required for inclusion. The primary endpoint was survival at 6 six-months from hrTMA diagnosis. Secondary endpoints were cumulative incidence of MODS 6 months after hrTMA diagnosis and 1-year posttransplant survival. Eculizumab dosing included intensive loading, induction, and maintenance phases for up to 24 weeks of therapy. All 21 evaluated study subjects had MODS. Primary and secondary study endpoints were met by demonstrating survival of 71% (P < .0001) 6 months after hrTMA diagnosis and 62% 1 year after transplant. Of fifteen survivors, 11 (73%) fully recovered organ function and are well. Our study demonstrates significant improvement in survival and recovery of organ function in hrTMA using an intensified eculizumab dosing and real time biomarker monitoring. This study serves as a benchmark for planning future studies that should focus on preventative measures or targeted therapy to be initiated prior to organ injury. This trial was registered at www.clinicaltrials.gov as #NCT03518203.

Molecular and Functional Characterization of Lymphoid Progenitor Subsets Reveals a Bipartite Architecture of Human Lymphopoiesis.

The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127- and CD127+ early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127- and CD127+ ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127- ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127+ ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis.

An acute immune response underlies the benefit of cardiac stem cell therapy.

Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day1,2, despite ongoing questions of efficacy and a lack of mechanistic understanding of the underlying biological effect3. The rationale for these cell therapy trials is derived from animal studies that show a modest but reproducible improvement in cardiac function in models of cardiac ischaemic injury4,5. Here we examine the mechanistic basis for cell therapy in mice after ischaemia-reperfusion injury, and find that-although heart function is enhanced-it is not associated with the production of new cardiomyocytes. Cell therapy improved heart function through an acute sterile immune response characterized by the temporal and regional induction of CCR2+ and CX3CR1+ macrophages. Intracardiac injection of two distinct types of adult stem cells, cells killed by freezing and thawing or a chemical inducer of the innate immune response all induced a similar regional accumulation of CCR2+ and CX3CR1+ macrophages, and provided functional rejuvenation to the heart after ischaemia-reperfusion injury. This selective macrophage response altered the activity of cardiac fibroblasts, reduced the extracellular matrix content in the border zone and enhanced the mechanical properties of the injured area. The functional benefit of cardiac cell therapy is thus due to an acute inflammatory-based wound-healing response that rejuvenates the infarcted area of the heart.

Biologic and Clinical Efficacy of LentiGlobin for Sickle Cell Disease.

BACKGROUND: Sickle cell disease is characterized by the painful recurrence of vaso-occlusive events. Gene therapy with the use of LentiGlobin for sickle cell disease (bb1111; lovotibeglogene autotemcel) consists of autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified ß-globin gene, which produces an antisickling hemoglobin, HbAT87Q. METHODS: In this ongoing phase 1-2 study, we optimized the treatment process in the initial 7 patients in Group A and 2 patients in Group B with sickle cell disease. Group C was established for the pivotal evaluation of LentiGlobin for sickle cell disease, and we adopted a more stringent inclusion criterion that required a minimum of four severe vaso-occlusive events in the 24 months before enrollment. In this unprespecified interim analysis, we evaluated the safety and efficacy of LentiGlobin in 35 patients enrolled in Group C. Included in this analysis was the number of severe vaso-occlusive events after LentiGlobin infusion among patients with at least four vaso-occlusive events in the 24 months before enrollment and with at least 6 months of follow-up. RESULTS: As of February 2021, cell collection had been initiated in 43 patients in Group C; 35 received a LentiGlobin infusion, with a median follow-up of 17.3 months (range, 3.7 to 37.6). Engraftment occurred in all 35 patients. The median total hemoglobin level increased from 8.5 g per deciliter at baseline to 11 g or more per deciliter from 6 months through 36 months after infusion. HbAT87Q contributed at least 40% of total hemoglobin and was distributed across a mean (±SD) of 85±8% of red cells. Hemolysis markers were reduced. Among the 25 patients who could be evaluated, all had resolution of severe vaso-occlusive events, as compared with a median of 3.5 events per year (range, 2.0 to 13.5) in the 24 months before enrollment. Three patients had a nonserious adverse event related or possibly related to LentiGlobin that resolved within 1 week after onset. No cases of hematologic cancer were observed during up to 37.6 months of follow-up. CONCLUSIONS: One-time treatment with LentiGlobin resulted in sustained production of HbAT87Q in most red cells, leading to reduced hemolysis and complete resolution of severe vaso-occlusive events. (Funded by Bluebird Bio; HGB-206 ClinicalTrials.gov number, NCT02140554.).

Axicabtagene Ciloleucel as Second-Line Therapy for Large B-Cell Lymphoma.

BACKGROUND: The prognosis of patients with early relapsed or refractory large B-cell lymphoma after the receipt of first-line chemoimmunotherapy is poor. METHODS: In this international, phase 3 trial, we randomly assigned, in a 1:1 ratio, patients with large B-cell lymphoma that was refractory to or had relapsed no more than 12 months after first-line chemoimmunotherapy to receive axicabtagene ciloleucel (axi-cel, an autologous anti-CD19 chimeric antigen receptor T-cell therapy) or standard care (two or three cycles of investigator-selected, protocol-defined chemoimmunotherapy, followed by high-dose chemotherapy with autologous stem-cell transplantation in patients with a response to the chemoimmunotherapy). The primary end point was event-free survival according to blinded central review. Key secondary end points were response and overall survival. Safety was also assessed. RESULTS: A total of 180 patients were randomly assigned to receive axi-cel and 179 to receive standard care. The primary end-point analysis of event-free survival showed that axi-cel therapy was superior to standard care. At a median follow-up of 24.9 months, the median event-free survival was 8.3 months in the axi-cel group and 2.0 months in the standard-care group, and the 24-month event-free survival was 41% and 16%, respectively (hazard ratio for event or death, 0.40; 95% confidence interval, 0.31 to 0.51; P<0.001). A response occurred in 83% of the patients in the axi-cel group and in 50% of those in the standard-care group (with a complete response in 65% and 32%, respectively). In an interim analysis, the estimated overall survival at 2 years was 61% in the axi-cel group and 52% in the standard-care group. Adverse events of grade 3 or higher occurred in 91% of the patients who received axi-cel and in 83% of those who received standard care. Among patients who received axi-cel, grade 3 or higher cytokine release syndrome occurred in 6% and grade 3 or higher neurologic events in 21%. No deaths related to cytokine release syndrome or neurologic events occurred. CONCLUSIONS: Axi-cel therapy led to significant improvements, as compared with standard care, in event-free survival and response, with the expected level of high-grade toxic effects. (Funded by Kite; ZUMA-7 ClinicalTrials.gov number, NCT03391466.).

Triplet Therapy, Transplantation, and Maintenance until Progression in Myeloma.

BACKGROUND: In patients with newly diagnosed multiple myeloma, the effect of adding autologous stem-cell transplantation (ASCT) to triplet therapy (lenalidomide, bortezomib, and dexamethasone [RVD]), followed by lenalidomide maintenance therapy until disease progression, is unknown. METHODS: In this phase 3 trial, adults (18 to 65 years of age) with symptomatic myeloma received one cycle of RVD. We randomly assigned these patients, in a 1:1 ratio, to receive two additional RVD cycles plus stem-cell mobilization, followed by either five additional RVD cycles (the RVD-alone group) or high-dose melphalan plus ASCT followed by two additional RVD cycles (the transplantation group). Both groups received lenalidomide until disease progression, unacceptable side effects, or both. The primary end point was progression-free survival. RESULTS: Among 357 patients in the RVD-alone group and 365 in the transplantation group, at a median follow-up of 76.0 months, 328 events of disease progression or death occurred; the risk was 53% higher in the RVD-alone group than in the transplantation group (hazard ratio, 1.53; 95% confidence interval [CI], 1.23 to 1.91; P<0.001); median progression-free survival was 46.2 months and 67.5 months. The percentage of patients with a partial response or better was 95.0% in the RVD-alone group and 97.5% in the transplantation group (P = 0.55); 42.0% and 46.8%, respectively, had a complete response or better (P = 0.99). Treatment-related adverse events of grade 3 or higher occurred in 78.2% and 94.2%, respectively; 5-year survival was 79.2% and 80.7% (hazard ratio for death, 1.10; 95% CI, 0.73 to 1.65). CONCLUSIONS: Among adults with multiple myeloma, RVD plus ASCT was associated with longer progression-free survival than RVD alone. No overall survival benefit was observed. (Funded by the National Heart, Lung, and Blood Institute and others; DETERMINATION ClinicalTrials.gov number, NCT01208662.).

How I treat transplant-eligible patients with myelofibrosis.

Despite the approval of Janus kinase inhibitors and novel agents for patients with myelofibrosis (MF), disease-modifying responses remain limited, and hematopoietic stem cell transplantation (HSCT) remains the only potentially curative treatment option. The number of HSCTs for MF continues to increase worldwide, but its inherent therapy-related morbidity and mortality limit its use for many patients. Furthermore, patients with MF often present at an older age, with cytopenia, splenomegaly, and severe bone marrow fibrosis, posing challenges in managing them throughout the HSCT procedure. Although implementation of molecular analyses enabled improved understanding of disease mechanisms and subsequently sparked development of novel drugs with promising activity, prospective trials in the HSCT setting are often lacking, making an evidence-based decision process particularly difficult. To illustrate how we approach patients with MF with respect to HSCT, we present 3 different clinical scenarios to capture relevant aspects that influence our decision making regarding indication for, or against, HSCT. We describe how we perform HSCT according to different risk categories and, furthermore, discuss our up-to-date approach to reduce transplant-related complications. Last, we show how to harness graft-versus-MF effects, particularly in the posttransplant period to achieve the best possible outcomes for patients.