Daniel C. Kirouac

The Systematic Production of Cells for Cell Therapies

Stem cells have emerged as the starting material of choice for bioprocesses to produce cells and tissues to treat degenerative, genetic, and immunological disease. Translating the biological properties and potential of stem cells into therapies will require overcoming significant cell-manufacturing and regulatory challenges. Bioprocess engineering fundamentals, including bioreactor design and process control, need to be combined with cellular systems biology principles to guide the development of next-generation technologies capable of producing cell-based products in a safe, robust, and cost-effective manner. The step-wise implementation of these bioengineering strategies will enhance cell therapy product quality and safety, expediting clinical development.

Rapid Expansion of Human Hematopoietic Stem Cells by Automated Control of Inhibitory Feedback Signaling

Clinical hematopoietic transplantation outcomes are strongly correlated with the numbers of cells infused. Anticipated novel therapeutic implementations of hematopoietic stem cells (HSCs) and their derivatives further increase interest in strategies to expand HSCs ex vivo. A fundamental limitation in all HSC-driven culture systems is the rapid generation of differentiating cells and their secreted inhibitory feedback signals. Herein we describe an integrated computational and experimental strategy that enables a tunable reduction in the global levels and impact of paracrine signaling factors in an automated closed-system process by employing a controlled fed-batch media dilution approach. Application of this system to human cord blood cells yielded a rapid (12-day) 11-fold increase of HSCs with self-renewing, multilineage repopulating ability. These results highlight the marked improvements that control of feedback signaling can offer primary stem cell culture and demonstrate a clinically relevant rapid and relatively low culture volume strategy for ex vivo HSC expansion.

Dynamic interaction networks in a hierarchically organized tissue

Intercellular (between cell) communication networks maintain homeostasis and coordinate regenerative and developmental cues in multicellular organisms. Despite the importance of intercellular networks in stem cell biology, their rules, structure and molecular components are poorly understood. Herein, we describe the structure and dynamics of intercellular and intracellular networks in a stem cell derived, hierarchically organized tissue using experimental and theoretical analyses of cultured human umbilical cord blood progenitors. By integrating high‐throughput molecular profiling, database and literature mining, mechanistic modeling, and cell culture experiments, we show that secreted factor‐mediated intercellular communication networks regulate blood stem cell fate decisions. In particular, self‐renewal is modulated by a coupled positive–negative intercellular feedback circuit composed of megakaryocyte‐derived stimulatory growth factors (VEGF, PDGF, EGF, and serotonin) versus monocyte‐derived inhibitory factors (CCL3, CCL4, CXCL10, TGFB2, and TNFSF9). We reconstruct a stem cell intracellular network, and identify PI3K, Raf, Akt, and PLC as functionally distinct signal integration nodes, linking extracellular, and intracellular signaling. This represents the first systematic characterization of how stem cell fate decisions are regulated non‐autonomously through lineage‐specific interactions with differentiated progeny.

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.

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.