Kirsi Sepponen1, Karolina Lundin1, Katri Knuus1, Pia Väyrynen1, Taneli Raivio2, Juha S Tapanainen1,3, Timo Tuuri1. 1. Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki 00029, Finland. 2. Department of Physiology, University of Helsinki, Helsinki 00014, Finland. 3. Department of Obstetrics and Gynecology, University Hospital of Oulu, University of Oulu, Medical Research Center Oulu and PEDEGO Research Unit, Oulu 90220, Finland.
Abstract
Context: Human gonads arise as a pair of epithelial ridges on the surface of intermediate mesoderm (IM)-derived mesonephros. Toxic environmental factors and mutations in various genes are known to disturb normal gonadal development, but because of a lack of suitable in vitro models, detailed studies characterizing the molecular basis of the observed defects have not been performed. Objective: To establish an in vitro method for studying differentiation of bipotential gonadal progenitors by using human embryonic stem cells (hESCs) and to investigate the role of bone morphogenetic protein (BMP) in gonadal differentiation. Design: We tested 17 protocols using activin A, CHIR-99021, and varying durations of BMP-7 and the BMP inhibitor dorsomorphin. Activation of activin A, WNT, and BMP pathways was optimized to induce differentiation. Setting: Academic research laboratory. Main Outcomes Measures: Cell differentiation, gene expression, and flow cytometry. Results: The two most efficient protocols consistently upregulated IM markers LHX1, PAX2, and OSR1 at days 2 to 4 and bipotential gonadal markers EMX2, GATA4, WT1, and LHX9 at day 8 of culture. The outcome depended on the combination of the duration, concentration, and type of BMP activation and the length of WNT signaling. Adjusting any of the parameters substantially affected the requirements for other parameters. Conclusions: We have established a reproducible protocol for directed differentiation of hESCs into bipotential gonadal cells. The protocol can be used to model early gonadal development in humans and allows further differentiation to mature gonadal somatic cells.
Context:Human gonads arise as a pair of epithelial ridges on the surface of intermediate mesoderm (IM)-derived mesonephros. Toxic environmental factors and mutations in various genes are known to disturb normal gonadal development, but because of a lack of suitable in vitro models, detailed studies characterizing the molecular basis of the observed defects have not been performed. Objective: To establish an in vitro method for studying differentiation of bipotential gonadal progenitors by using human embryonic stem cells (hESCs) and to investigate the role of bone morphogenetic protein (BMP) in gonadal differentiation. Design: We tested 17 protocols using activin A, CHIR-99021, and varying durations of BMP-7 and the BMP inhibitor dorsomorphin. Activation of activin A, WNT, and BMP pathways was optimized to induce differentiation. Setting: Academic research laboratory. Main Outcomes Measures: Cell differentiation, gene expression, and flow cytometry. Results: The two most efficient protocols consistently upregulated IM markers LHX1, PAX2, and OSR1 at days 2 to 4 and bipotential gonadal markers EMX2, GATA4, WT1, and LHX9 at day 8 of culture. The outcome depended on the combination of the duration, concentration, and type of BMP activation and the length of WNT signaling. Adjusting any of the parameters substantially affected the requirements for other parameters. Conclusions: We have established a reproducible protocol for directed differentiation of hESCs into bipotential gonadal cells. The protocol can be used to model early gonadal development in humans and allows further differentiation to mature gonadal somatic cells.
Authors: K Lundin; K Sepponen; P Väyrynen; X Liu; D A Yohannes; M Survila; B Ghimire; J Känsäkoski; S Katayama; J Partanen; S Vuoristo; P Paloviita; N Rahman; T Raivio; K Luiro; I Huhtaniemi; M Varjosalo; T Tuuri; J S Tapanainen Journal: Mol Hum Reprod Date: 2022-04-29 Impact factor: 4.518
Authors: Ingrid M Knarston; Svenja Pachernegg; Gorjana Robevska; Irene Ghobrial; Pei Xuan Er; Elizabeth Georges; Minoru Takasato; Alexander N Combes; Anne Jørgensen; Melissa H Little; Andrew H Sinclair; Katie L Ayers Journal: Stem Cell Reports Date: 2020-11-19 Impact factor: 7.765