Development and Validation of a Rex1-RFP Potency Activity Reporter Assay That Quantifies Stress-Forced Potency Loss in Mouse Embryonic Stem Cells

Abstract

Assays for embryonic stem cells (ESCs) of the blastocyst are needed to quantify stress-induced decreases of potent subpopulations. High-throughput screens (HTSs) of stressed ESCs quantify embryonic stress, diminishing laboratory animal needs. Normal or stress-induced ESC differentiation is marked by Rex1 potency factor loss. Potency reporter ESC assays were developed, using low-stress techniques to create transgenic ESCs. Rex1 and Oct4 promoters drove RFP and green fluorescent protein (GFP) expression, respectively. Lentivirus infection and fluorescence-activated cell sorting selection of ESCs obviated the need for stressful electroporation and antibiotic selection, respectively. We showed using immunoblots, microscopic analysis, flow cytometry, and fluorescence microplate reader that the response to stress of potency-reporter ESCs is similar to parental ESCs assayed by biochemical means. Stress caused a dose-dependent decrease in bright Rex1-RFP⁺ ESCs and increase in Rex1 dim ESCs. At highest stress, ∼20% of bright Rex1-RFP cells are lost coinciding with a 2.8-fold increase in Rex1-RFP dim cells that approach 20%. This conversion of bright to dim cells tested by flow cytometry is commensurate with about 60% loss in fluorescence measured by microplate reader. Dose-dependent stress-induced Rex1-RFP and endogenous Rex1 protein decreases are similar. The data show that Rex1 reporter ESCs accurately report stress in a microplate reader-based HTS. The increasing dim Rex1 subpopulation size is balanced by the decreasing total ESC number during culture at multiple sorbitol doses. This is consistent with previous observations that stress forces potency decrease and differentiation increase to compensate for stress-induced diminished stem cell population growth.