Creation And Optimization Of A Method For Inducing Differentiation Of Human Embryonic Stem Cells Into Parathyroid-like Cells

Objective: Hypoparathyroidism is an endocrine disease characterized by low serum calcium concentration,which is caused by insufficient or absent secretion of parathyroid hormone(PTH).The aim of this study was to develop a new protocol for inducing human embryonic stem cells(h ESCs)to differentiate into parathyroid like cells in vitro,and to provide a new cell therapy for patients with congenital and secondary hypoparathyroidism.Methods: By mimicking the embryonic development of human parathyroid glands,we proposed a new differentiation hypothesis on the basis of previous studies,that is,according to four stages of differentiation: definitive endoderm,foregut endoderm,pharyngeal pouch endoderm and parathyroid cells,h ESCs were gradually differentiated into human embryonic stem cells-derived parathyroid like cells(h ESC-PT).At each differentiation stage,the m RNA and protein expression levels of differentiation markers were checked by PCR and immunofluorescence assays,the differentiation rates were also quantitatively analyzed.Bulk RNA sequencing analysis was performed on h ESCs and h ESC-PT to follow the changes in cellular transcription levels before and after differentiation.The PTH secretion function of h ESC-PT was evaluated by enzyme linked immunosorbent assay(ELISA).In order to optimize the differentiation protocol and further improve the differentiation rate of PTH-positive cells,we designed lentivirus transfection and performed organoid experiments.GCM2 is a key gene in parathyroid embryonic development.We successfully established an h ESC cell line for stably overexpressing GCM2 with lentivirus transfection and induced its directed differentiation for examining the role of GCM2.Parathyroid organoids were also constructed to evaluate how a three-dimensional culturing environment affects the differentiation of h ESC-PT cells.Results: HESCs could differentiate into h ESC-PT by following the four-stage differentiation protocol.99.33% of the cells co-expressed SOX17 and FOXA2 when the first stage was completed.96.51% cells co-expressed SOX2 and FOXA2 when the second stage was completed.When the third stage was completed,30.89% cells expressed HOXA3 protein and 27.90% cells expressed EYA1.16.96% of the h ESC-PT cells expressed PTH when the fourth stage was completed.In addition,GCM2,Ca SR,and CHGA were also detected in h ESC-PT cells.Bulk RNA sequencing results showed that the expression of parathyroid related genes was significantly up-regulated in h ESC-PT cells.h ESC-PT cells could secrete PTH,and low calcium concentration could promote the secretion of PTH.Lentiviral transfection experiment suggested that GCM2 protein promoted cell maturation.Compared with the two-dimensional culture,construction of parathyroid organoids promoted the differentiation of h ESC-PT cells,which could increase the PTH secretion seen in the supernatant.Conclusions: In this study,a novel approach was developed to induce the differentiation of human embryonic stem cells into h ESC-PT cells in vitro.Then the protocol was optimized through the GCM2 protein overexpression and organoid construction,which laid a solid foundation for using stem cell replacement therapy toward congenital and secondary hypoparathyroidism.