Background: Amniotic fluid-derived mesenchymal stromal cells (AFMSCs) are promising stem cells for regeneration medicine. However, AFMSCs isolated at different stages of pregnancy have different biological characteristics, and the therapeutic effects can differ in vivo and in vitro. The mechanisms underlying these differences have not been defined. Methods: Bioinformatics analysis of the AFMSC transcriptome identified Chrdl1 as one of the differentially expressed genes. We evaluated the effects of Chrdl1 overexpression or knockdown on the proliferation and migration of AFMSCs. Target prediction was performed using miRanda software to identify the upstream microRNA of Chrdl1. The interaction between Chrdl1 mRNA and its upstream microRNA was evaluated using a dual-luciferase reporter gene assay. Results: Chrdl1 was expressed at lower levels in AFMSCs derived from the early stages of pregnancy. It could suppress AFMSC proliferation and migration. miR-532-3p promoted AFMSC proliferation and migration by targeting the 3' UTR of Chrdl1 and downregulating its expression. (c) 2020 Elsevier Inc. All rights reserved.