Engineering the haemogenic niche mitigates endogenous inhibitory signals and controls pluripotent stem cell-derived blood emergence

Abstract

Efforts to recapitulate haematopoiesis, a process guided by spatial and temporal inductive signals, to generate haematopoietic progenitors from human pluripotent stem cells (hPSCs) have focused primarily on exogenous signalling pathway activation or inhibition. Here we show haemogenic niches can be engineered using microfabrication strategies by micropatterning hPSC-derived haemogenic endothelial (HE) cells into spatially-organized, size-controlled colonies. CD34+VECAD+ HE cells were generated with multi-lineage potential in serum-free conditions and cultured as size-specific haemogenic niches that displayed enhanced blood cell induction over non-micropatterned cultures. Intra-colony analysis revealed radial organization of CD34 and VECAD expression levels, with CD45+ blood cells emerging primarily from the colony centroid area. We identify the induced interferon gamma protein (IP-10)/p-38 MAPK signalling pathway as the mechanism for haematopoietic inhibition in our culture system. Our results highlight the role of spatial organization in hPSC-derived blood generation, and provide a quantitative platform for interrogating molecular pathways that regulate human haematopoiesis.

A stepwise model of Reaction-Diffusion and Positional-Information governs self-organized human peri-gastrulation-like patterning

“…Because human embryos are not typically available for direct investigation, studying human gastrulation-associated fate patterning requires in vitro platforms that allow robust simulation and investigation of the signaling programs that initiate and drive gastrulation-like events. We and others have previously used micro-patterning technologies to control human (h)PSC colony geometry, demonstrating improved cell response consistency than is achieved in conventional adherent cultures ( Nazareth et al, 2013 ; Peerani et al, 2007 ; Bauwens et al, 2008 , 2011 ; Alom Ruiz and Chen, 2007 ; Stevens et al, 2013 ; Ma et al, 2015 ; Rahman et al, 2017 ). These studies highlight that control of endogenous signaling profiles, cell-cell contact, and mechanical forces are crucial to regulate cell fate and spatial tissue organization robustly.…”

Stereotypical architecture of the stem cell niche is spatiotemporally established by miR-125-dependent coordination of Notch and steroid signaling

“…Stem cells are necessary for the formation and maintenance of any organ. They can sustain their stemness only when located within specialized stem cell niches, which provide the lifelong support, a molecular address and tissue-specific milieu for adult stem cells ( Mathieu et al, 2013 ; Rahman et al, 2017 ; Rodríguez-Colman et al, 2017 ). However, our current knowledge about the processes governing stem cell niche assembly in any organism is very limited.…”

Context-explorer: Analysis of spatially organized protein expression in high-throughput screens

“…Members of our group regularly use CE for colony level analyses as evidenced in previously published and ongoing studies. (Nazareth et al, 2013, Rahman et al, 2017. To further broaden the utility and applications of the software, there are built-in visualizations to assist with fluorescent intensity thresholding and pattern fidelity assessment, and interface components to assign wells to treatment groups.…”