A New FACS Approach Isolates hESC Derived Endoderm Using Transcription Factors

Yuqiong Pan,Wing Hung Wong,Zhengqing Ouyang,Julie C Baker,Maurizio Pesce

doi:10.1371/journal.pone.0017536

Abstract

We show that high quality microarray gene expression profiles can be obtained following FACS sorting of cells using combinations of transcription factors. We use this transcription factor FACS (tfFACS) methodology to perform a genomic analysis of hESC-derived endodermal lineages marked by combinations of SOX17, GATA4, and CXCR4, and find that triple positive cells have a much stronger definitive endoderm signature than other combinations of these markers. Additionally, SOX17+ GATA4+ cells can be obtained at a much earlier stage of differentiation, prior to expression of CXCR4+ cells, providing an important new tool to isolate this earlier definitive endoderm subtype. Overall, tfFACS represents an advancement in FACS technology which broadly crosses multiple disciplines, most notably in regenerative medicine to redefine cellular populations.

Highlights

Cells in the developing embryo undergo step-wise progression toward particular fates
The results showed comparable staining for fixed and unfixed cells for the cell surface markers we have used in our experiments, including CXCR4 (Fig. S1A)
HESCs can differentiate into endodermal cells by dosing with high levels of the NODAL signaling pathway, but it remains unknown whether this differentiation results in several endodermal cell sub-types or a single homogeneous population

Summary

Introduction

Cells in the developing embryo undergo step-wise progression toward particular fates. Understanding the details of this progression program is dependent upon marking and identifying the emerging cellular populations. The ability to classify other developmental lineages in this rigorous manner would be a significant advance for developmental biology and for regenerative medicine, which greatly depends upon understanding and selecting pure populations of precise cellular types. Comprehensive cell surface markers have been difficult to identify in embryonic lineages, and teasing apart the stepwise progression of these lineages using Fluorescence Activated Cell Sorting (FACS) has remained difficult. Cell surface markers have not been well characterized in these emerging cell types, transcription factors are known to mark cellular lineages [4,5,6,7,8]. To date using nuclear proteins to examine cellular phenotypes has not been feasible due to limitations in technology [9]

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Conclusion