Abstract

Culture products of stem cell differentiation and somatic cell reprogramming, as well as native tissues, exhibit high cell type heterogeneity. Specific cell surface markers are often unknown, hampering molecular analysis of defined subpopulations. We report a method to obtain high-quality, global gene expression profiles of primary human and hESC-derived cells purified by fluorescence-activated cell sorting (FACS) using intracellular/cytoplasmic markers.  The method enabled, for the first time, the discovery of the molecular signatures of primary human fetal and adult pancreatic beta-cells FACS-purified from other pancreatic cell types. These included novel and known beta-cell factors. For instance, both fetal and adult beta-cells were enriched for the transcription factors Pdx1 and Nkx6-1, while only fetal beta-cells were enriched for the earlier developmental regulators FoxA2 and Pax4, and adult beta-cells preferentially expressed the maturity markers MafA and Ucn3. Subsequently, we compared these beta-cell transcriptomes to that of insulin-positive cells derived from genetically unmodified hESCs. Human ESC-derived insulin-positive cells were more similar to fetal beta-cells than to adult β-cells, but also lacked critical insulin processing/secretion machinery and expressed many genes characteristic of other endodermal cell types, Additionally, insulin-positive cells derived from multiple pluripotent stem cell lines were highly similar. We suggest that probing the differences in genes and pathways between hESC-derived, fetal, and adult beta-cells will provide heuristics for improving directed differentiation protocols and generating functionally mature beta-cells that could ameliorate type 1 diabetes upon transplantation. More generally, this approach holds broad potential for comparing specifically defined cell populations from in vitro stem cell-derived cultures and native human tissues.