Integrated Multi-Stage Tissue on a Chip Generation from Human Pluripotent Stem Cells

The development of human organs-on-chips, in which the microscale engineering technologies are combined with cultured human cells to recapitulate whole living organ microenvironment, offers a unique opportunity to study human physiology and pathophysiology in an organ-specific context^1,2,3. Recently, successful examples of organs-on-chips development have been provided^4,5,6,7, mainly using primary animal cells and, in few cases, primary human cells. The possibility of developing direct organogenesis-on-chip from human pluripotent stem cells (hPSCs) could overcome the limited availability of human primary cells, such as hepatocytes and cardiomyocytes. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) open a wide perspective for multi-organ generation on a chip. Integrated functional hPSCs differentiation on a chip has not yet been developed.

In this work, we demonstrate how to control stem cell expansion, selective germ layer commitment and derive functional tissue-specific cells on a chip from both hESCs and hiPSCs through a multi-stage microfluidic-based technology.

Experimentally, we showed that a discontinuous periodic medium delivery with stage-dependent frequency, f, (number of cycles of medium change per day) is an effective strategy for modulating stem cell niche specification in vitro, ensuring optimal delivery of exogenous factors and removal of endogenous cell-secreted factors. HPSCs homogeneously express pluripotency markers, such as Oct4, Sox2 and Tra1-60, along the microfluidic channel, as revealed by immunostaining. QRT-PCR analysis of Oct4 and Nanog after 5 days of culture, showed an optimal frequency of f=2d^-1 and a significant three-fold higher expression compared to lower and higher frequencies was observed.

HPSCs were induced to spontaneously differentiate within the microfluidic platform and a frequency-dependent germ layer enrichment was observed. Compared to standard culture in Petri dish, we obtained a significant increase in ectoderm markers OTX2 and TUBB3 expression by adopting f=1d^-1 and a dramatic reduction with f=8d^-1, confirming that ectoderm commitment is mainly driven by endogenous factors accumulation. Conversely, by adopting f=8d^-1, we observed a significant increase of meso-endoderm T and GATA4 markers and early endoderm FOXA2, EOMES and AFP markers expression. More selective germ layer commitment was successfully obtained following specific differentiation protocols and an optimal f for each germ layer commitment was established.

Cardiac cells on a chip were derived in 15 days of frequency-dependent multi-stage differentiation protocol, consisting on mesoderm induction, early cardiac commitment and functional maturation. 60% Troponin-T positive cells showed defined sarcomeric organization, spontaneous contractile activity, excitation contraction coupling up to 2 Hz and proper calcium dynamics. Cells were also responsive for caffeine and verapamil stimulation.

Similarly, hepatic-like cells on a chip were obtained, starting from endoderm commitment, up to hepatic maturation. Cells express cytokeratin 18, CYP-3A, which is the most relevant marker for drug toxicity assesment, and albumin. Cells are also responsive for indocyanine green digestion, glycogen storage (75% of total cells) and show 40% increase in albumin secretion, compared to static conditions.

Functionally differentiated cells derived in the microfluidic channels can be directly used for dynamic multi-parametric and large-scale drug screening or for developing micro-engineered human organ models, overcoming issues related to the limited availability of human primary cell sources.

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