Abstract
In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis, but the underlying processes are poorly understood and controlled. Here we show that modulating the bulk cell density (BCD: cell number per culture volume) deterministically alters anteroposterior patterning of primitive streak (PS)-like priming. The BCD in conjunction with the chemical WNT pathway activator CHIR99021 results in distinct paracrine microenvironments codifying hPSCs towards definitive endoderm, precardiac or presomitic mesoderm within the first 24 h of differentiation, respectively. Global gene expression and secretome analysis reveals that TGFß superfamily members, antagonist of Nodal signalling LEFTY1 and CER1, are paracrine determinants restricting PS progression. These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. By demonstrating a decisive, functional role of the BCD, we show its utility as a method to control lineage-specific differentiation. Furthermore, these findings have profound consequences for inter-experimental comparability, reproducibility, bioprocess optimization and scale-up.
Highlights
In vitro differentiation of human pluripotent stem cells recapitulates early aspects of human embryogenesis, but the underlying processes are poorly understood and controlled
In 1 ml medium per well (BCD: 1 Â 106 cells per ml) higher CHIR concentrations were concomitant with increased green fluorescent protein (GFP) expression (CHIR optimum at 15 mM; Fig. 1b and Supplementary Fig. 1e)
Efficient cardiomyogenesis was achieved at CHIR concentrations ranging from 5 to 15 mM, notably overlapping with 3–5 mM used for definitive endoderm 41–43 and 6–10 mM for presomitic mesoderm (PSM) derivatives[27,28]
Summary
In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis, but the underlying processes are poorly understood and controlled. Global gene expression and secretome analysis reveals that TGF superfamily members, antagonist of Nodal signalling LEFTY1 and CER1, are paracrine determinants restricting PS progression These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. Human pluripotent stem cells (hPSCs), including embryonic (hESCs) and induced pluripotent stem cells, provide an attractive model to study early aspects of human embryogenesis in vitro, which are not assessable in vivo. Due to their growth and differentiation potential, hPSCs constitute a well-characterized, generally unlimited cell source for the mass generation of lineage- and possibly patient-specific progenies. It is well established that the spatial patterning during gastrulation in mouse embryos is under the control of the Activin-Nodal, BMP and WNT pathways, which can be manipulated to direct mouse and hESCs into derivatives of all three germ layers[10,11,12,13]
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