The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.

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.

Co-culture strategies are foundational in cell biology. These systems, which serve as mimics of in vivo tissue niches, are typically poorly defined in terms of cell ratios, local cues and supportive cell-cell interactions. In the stem cell niche, the ability to screen cell-cell interactions and identify local supportive microenvironments has a broad range of applications in transplantation, tissue engineering and wound healing. We present a microfluidic platform for the high-throughput generation of hydrogel microbeads for cell co-culture. Encapsulation of different cell populations in microgels was achieved by introducing in a microfluidic device two streams of distinct cell suspensions, emulsifying the mixed suspension, and gelling the precursor droplets. The cellular composition in the microgels was controlled by varying the volumetric flow rates of the corresponding streams. We demonstrate one of the applications of the microfluidic method by co-encapsulating factor-dependent and responsive blood progenitor cell lines (MBA2 and M07e cells, respectively) at varying ratios, and show that in-bead paracrine secretion can modulate the viability of the factor dependent cells. Furthermore, we show the application of the method as a tool to screen the impact of specific growth factors on a primary human heterogeneous cell population. Co-encapsulation of IL-3 secreting MBA2 cells with umbilical cord blood cells revealed differential sub-population responsiveness to paracrine signals (CD14+ cells were particularly responsive to locally delivered IL-3). This microfluidic co-culture platform should enable high throughput screening of cell co-culture conditions, leading to new strategies to manipulate cell fate.

We have integrated gene expression profiling with database and literature mining, mechanistic modeling, and cell culture experiments to identify intercellular and intracellular networks regulating blood stem cell self-renewal.Blood stem cell fate in vitro is regulated non-autonomously 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).The antagonistic signals converge in a core intracellular network focused around PI3K, Raf, PLC, and Akt.Model simulations enable functional classification of the novel endogenous ligands and signaling molecules.