Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

Christian Unger,Tom J.R. Frith,Martin I. Garcia-Castro,Peter W. Andrews,Ana Marin Navarro,James O.S. Hackland,Oliver Thompson

doi:10.1016/j.stemcr.2017.08.008

Abstract

SummaryDefects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.

Highlights

Neural crest, a transient, migratory tissue arising during vertebrate development, is first observed in the human at Carnegie stage 9 (O’Rahilly and Muller, 2007)
Human neural crest expresses many of the markers found in other vertebrates, including SOX10, PAX3, TFAP2a, and p75 (Betters et al, 2010), and when human pluripotent stem cells (hPSCs) are differentiated into neural crest they share this expression profile and can generate neural crestderived lineages in vitro (Menendez et al, 2011; Mica et al, 2013)
We describe a fully defined and xeno-free system for the differentiation of hPSCs into neural crest and show that an optimum level of BMP activity is required for neural crest induction, whereas higher or lower levels lead to the induction of genes associated with non-neural or neural identity, respectively

Summary

SUMMARY

Defects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. The protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can affect the outcome of differentiation protocols

INTRODUCTION

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DISCUSSION

Conclusion

EXPERIMENTAL PROCEDURES