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Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

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Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells.

Henning Kempf^1,2, Ruth Olmer^1,2,3, Alexandra Haase^1,2

¹Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.

Nature Communications

|December 10, 2016

Summary

Controlling human pluripotent stem cell (hPSC) differentiation involves modulating bulk cell density (BCD). This method guides hPSCs toward specific mesoderm types, improving reproducibility and scale-up for developmental biology research.

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Area of Science:

Developmental Biology
Stem Cell Biology
Biotechnology

Background:

Human pluripotent stem cell (hPSC) differentiation models early human embryogenesis.
Understanding and controlling these processes, particularly primitive streak (PS) patterning, remains challenging.

Purpose of the Study:

To investigate the role of bulk cell density (BCD) in controlling hPSC differentiation.
To establish a method for deterministic lineage specification of hPSCs.

Main Methods:

Modulation of BCD in combination with WNT pathway activation (CHIR99021).
Global gene expression and secretome analysis.
Analysis of paracrine signaling factors like TGFß superfamily members, LEFTY1, and CER1.

Main Results:

BCD deterministically alters anteroposterior patterning of PS-like priming in hPSCs.
Specific BCDs combined with CHIR99021 induce definitive endoderm, precardiac, or presomitic mesoderm within 24 hours.
Paracrine factors, including LEFTY1 and CER1, restrict PS progression and influence lineage commitment.

Conclusions:

BCD is a critical, controllable factor in directing hPSC lineage specification.
This finding offers a method to control lineage-specific differentiation, enhancing experimental reproducibility and bioprocess scale-up.