SummaryReproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.

Embryonic stem cells progress very rapidly through the cell cycle, allowing limited time for cell cycle regulatory circuits that typically function in somatic cells. Mechanisms that inhibit cell cycle progression upon DNA damage are of particular importance, as their malfunction may contribute to the genetic instability observed in human embryonic stem cells (hESCs). In this study, we exposed undifferentiated hESCs to DNA-damaging ultraviolet radiation-C range (UVC) light and examined their progression through the G1/S transition. We show that hESCs irradiated in G1 phase undergo cell cycle arrest before DNA synthesis and exhibit decreased cyclin-dependent kinase two (CDK2) activity. We also show that the phosphatase Cdc25A, which directly activates CDK2, is downregulated in irradiated hESCs through the action of the checkpoint kinases Chk1 and/or Chk2. Importantly, the classical effector of the p53-mediated pathway, protein p21, is not a regulator of G1/S progression in hESCs. Taken together, our data demonstrate that cultured undifferentiated hESCs are capable of preventing entry into Sphase by activating the G1/S checkpoint upon damage to their genetic complement.

Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53-p21 axis of the G1/ S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA (miRNA) pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of miRNAs, including hESCspecific miRNAs such as those of the miR-302 family, miR-371-372 family, or C19MC miRNA cluster. Most importantly, we show that the hESC-enriched miRNA family miR-302 (miR-302a, miR-302b, miR-302c, and miR-302d) directly contributes to regulation of p21 expression in hESCs and, thus, demonstrate a novel function for miR-302s in hESCS. The described mechanism elucidates the role of miRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage. STEM CELLS