Gerard J. Madlambayan

Cell–cell interaction networks regulate blood stem and progenitor cell fate

Communication networks between cells and tissues are necessary for homeostasis in multicellular organisms. Intercellular (between cell) communication networks are particularly relevant in stem cell biology, as stem cell fate decisions (self‐renewal, proliferation, lineage specification) are tightly regulated based on physiological demand. We have developed a novel mathematical model of blood stem cell development incorporating cell‐level kinetic parameters as functions of secreted molecule‐mediated intercellular networks. By relation to quantitative cellular assays, our model is capable of predictively simulating many disparate features of both normal and malignant hematopoiesis, relating internal parameters and microenvironmental variables to measurable cell fate outcomes. Through integrated in silico and experimental analyses, we show that blood stem and progenitor cell fate is regulated by cell–cell feedback, and can be controlled non‐cell autonomously by dynamically perturbing intercellular signalling. We extend this concept by demonstrating that variability in the secretion rates of the intercellular regulators is sufficient to explain heterogeneity in culture outputs, and that loss of responsiveness to cell–cell feedback signalling is both necessary and sufficient to induce leukemic transformation in silico.

Leukemia regression by vascular disruption and antiangiogenic therapy.

Acute myelogenous leukemias (AMLs) and endothelial cells depend on each other for survival and proliferation. Monotherapy antivascular strategies such as targeting vascular endothelial growth factor (VEGF) has limited efficacy in treating AML. Thus, in search of a multitarget antivascular treatment strategy for AML, we tested a novel vascular disrupting agent, OXi4503, alone and in combination with the anti-VEGF antibody, bevacizumab. Using xenotransplant animal models, OXi4503 treatment of human AML chloromas led to vascular disruption in leukemia cores that displayed increased leukemia cell apoptosis. However, viable rims of leukemia cells remained and were richly vascular with increased VEGF-A expression. To target this peripheral reactive angiogenesis, bevacizumab was combined with OXi4503 and abrogated viable vascular rims, thereby leading to enhanced leukemia regression. In a systemic model of primary human AML, OXi4503 regressed leukemia engraftment alone and in combination with bevacizumab. Differences in blood vessel density alone could not account for the observed regression, suggesting that OXi4503 also exhibited direct cytotoxic effects on leukemia cells. In vitro analyses confirmed this targeted effect, which was mediated by the production of reactive oxygen species and resulted in apoptosis. Together, these data show that OXi4503 alone is capable of regressing AML by a multitargeted mechanism and that the addition of bevacizumab mitigates reactive angiogenesis.

Bone marrow stem and progenitor cell contribution to neovasculogenesis is dependent on model system with SDF-1 as a permissive trigger

Adult bone marrow (BM) contributes to neovascularization in some but not all settings, and reasons for these discordant results have remained unexplored. We conducted novel comparative studies in which multiple neovascularization models were established in single mice to reduce variations in experimental methodology. In different combinations, BM contribution was detected in ischemic retinas and, to a lesser extent, Lewis lung carcinoma cells, whereas B16 melanomas showed little to no BM contribution. Using this spectrum of BM contribution, we demonstrate the necessity for site-specific expression of stromal-derived factor-1alpha (SDF-1alpha) and its mobilizing effects on BM. Blocking SDF-1alpha activity with neutralizing antibodies abrogated BM-derived neovascularization in lung cancer and retinopathy. Furthermore, secondary transplantation of single hematopoietic stem cells (HSCs) showed that HSCs are a long-term source of neovasculogenesis and that CD133(+)CXCR4(+) myeloid progenitor cells directly participate in new blood vessel formation in response to SDF-1alpha. The varied BM contribution seen in different model systems is suggestive of redundant mechanisms governing postnatal neovasculogenesis and provides an explanation for contradictory results observed in the field.

Myxoma virus targets primary human leukemic stem and progenitor cells while sparing normal hematopoietic stem and progenitor cells.

Myxoma virus targets primary human leukemic stem and progenitor cells while sparing normal hematopoietic stem and progenitor cells

Functional integration of acute myeloid leukemia into the vascular niche

Vascular endothelial cells are a critical component of the hematopoietic microenvironment that regulates blood cell production. Recent studies suggest the existence of functional cross-talk between hematologic malignancies and vascular endothelium. Here we show that human acute myeloid leukemia (AML) localizes to the vasculature in both patients and in a xenograft model. A significant number of vascular tissue-associated AML cells (V-AML) integrate into vasculature in vivo and can fuse with endothelial cells. V-AML cells acquire several endothelial cell-like characteristics, including the upregulation of CD105, a receptor associated with activated endothelium. Remarkably, endothelial-integrated V-AML shows an almost fourfold reduction in proliferative activity compared with non-vascular-associated AML. Primary AML cells can be induced to downregulate the expression of their hematopoietic markers in vitro and differentiate into phenotypically and functionally defined endothelial-like cells. After transplantation, these leukemia-derived endothelial cells are capable of giving rise to AML. These novel functional interactions between AML cells and normal endothelium along with the reversible endothelial cell potential of AML suggest that vascular endothelium may serve as a previously unrecognized reservoir for AML.

Umbilical cord-derived stem cells for tissue therapy: current and future uses

Organ and tissue transplants provide a means to correct disease but are limited, mostly owing to the lack of available donor tissue. Tissue matching and speed of procurement are important parameters that must be met for a successful graft, however the lack of available donors leads to expanding waiting lists and suboptimal human leukocyte antigen-matching, often leading to reduced transplant success. The discovery of embryonic stem cells and tissue-specific stem cells has provided hope for many patients. Stem cell-based therapy has provided possible new sources of human leukocyte antigen-matched tissue but, before routine clinical application of stem cells becomes a reality, many obstacles must first be overcome. Focusing on umbilical cord blood cells, we discuss some of the challenges that stem cell therapy faces, including obtaining clinically relevant numbers of stem cells and the ability of stem cells to provide for permanent engraftment of multiple tissue types. We discuss possible solutions to these problems, such as in vitro stem cell expansion and the differentiation potential of tissue-specific stem cells.

The AC133+CD38−, but not the rhodamine-low, phenotype tracks LTC-IC and SRC function in human cord blood ex vivo expansion cultures

Phenotypic markers associated with human hematopoietic stem cells (HSCs) were developed and validated using uncultured cells. Because phenotype and function can be dissociated during culture, better markers to prospectively track and isolate HSCs in ex vivo cultures could be instrumental in advancing HSC-based therapies. Using an expansion system previously shown to increase hematopoietic progenitors and SCID-repopulating cells (SRCs), we demonstrated that the rhodamine-low phenotype was lost, whereas AC133 expression was retained throughout culture. Furthermore, the AC133(+)CD38(-) subpopulation was significantly enriched in long-term culture-initiating cells (LTC-IC) and SRCs after culture. Preculture and postculture analysis of total nucleated cell and LTC-IC number, and limiting dilution analysis in NOD/SCID mice, showed a 43-fold expansion of the AC133(+)CD38(-) subpopulation that corresponded to a 7.3-fold and 4.4-fold expansion of LTC-ICs and SRCs in this subpopulation, respectively. Thus, AC133(+)CD38(-) is an improved marker that tracks and enriches for LTC-IC and SRC in ex vivo cultures.

Acute myeloid leukemia targeting by myxoma virus in vivo depends on cell binding but not permissiveness to infection in vitro

Some oncolytic viruses, such as myxoma virus (MYXV), can selectively target malignant hematopoietic cells, while sparing normal hematopoietic cells. This capacity for discrimination creates an opportunity to use oncolytic viruses as ex vivo purging agents of autologous hematopoietic cell grafts in patients with hematologic malignancies. However, the mechanisms by which oncolytic viruses select malignant hematopoietic cells are poorly understood. In this study, we investigated how MYXV specifically targets human AML cells. MYXV prevented chloroma formation and bone marrow engraftment of two human AML cell lines, KG-1 and THP-1. The reduction in human leukemia engraftment after ex vivo MYXV treatment was dose-dependent and required a minimum MOI of 3. Both AML cell lines demonstrated MYXV binding to leukemia cell membranes following co-incubation: however, evidence of productive MYXV infection was observed only in THP-1 cells. This observation, that KG-1 can be targeted in vivo even in the absence of in vitro permissive viral infection, contrasts with the current understanding of oncolytic virotherapy, which assumes that virus infection and productive replication is a requirement. Preventing MYXV binding to AML cells with heparin abrogated the purging capacity of MYXV, indicating that binding of infectious virus particles is a necessary step for effective viral oncolysis. Our results challenge the current dogma of oncolytic virotherapy and show that in vitro permissiveness to an oncolytic virus is not necessarily an accurate predictor of oncolytic potency in vivo.

Marrow cell therapies for cardiovascular diseases

The nascent field of regenerative medicine has taken root in cardiovascular disease. Preclinical data demonstrating hemangioblast potential of marrow cells and cardioprotective effects of growth factors served as the basis for numerous early phase clinical trials. With the first wave of safety and efficacy trials complete, much is still unknown regarding optimal cell dose and type, timing of injection, route of administration, mechanisms of action, and achievable response measures. The next generation of studies will aim to answers these questions and make way for cellular therapies that result in effective cardiac repair.