Differentiation roadmap of embryonic Sertoli cells derived from mouse embryonic stem cells

Abstract

Background: Embryonic Sertoli cells (eSCs) play an important role in sex determination and in male gonad development which makes them a very useful cell type for therapeutic applications. However, the deriving mechanism of Sertoli cells has been unclear and challenging to create a large number of quality eSCs. Therefore, this study aimed to create the eSCs induced from mouse embryonic stem (mES) cells by regulating defined factors and to explore the relevant regulatory mechanism.

Methods: Six inducing factors, Sry, Sox9, SF1, WT1, GATA4, and Dmrt1, were respectively transduced into mES cells by lentiviral infection according to the experimental design. The test groups were identified by development stage-specific markers, AMH, Emx2, SF1, and FasL, using flow cytometry. Induced eSCs were determined by FasL and AMH biomarkers under immunofluorescence, immunocytochemistry, and flow cytometry. Moreover, the pluripotency markers, gonad development-related markers, epithelial markers and mesenchymal markers in test groups were transcriptionally determined by qPCR.

Results: In this study, the co-overexpression of all the six factors effectively produced a large population of eSCs from mES cells in 35 days of culturing. These eSCs were capable of forming tubular-like and ring-like structures with functional performance. The results of flow cytometry indicated that the upregulation of GATA4 and WT1 contributed to the growth of somatic cells in the coelomic epithelium regarded as the main progenitor cells of eSCs. Whereas, SF1 facilitated the development of eSC precursor cells, and Sry and Sox9 promoted the determination of male development. Moreover, the overexpression of Dmrt1 was essential for the maintenance of eSCs and some of their specific surface biomarkers such as FasL. The cellular morphology, biomarker identification, and transcriptomic analysis aided in exploring the regulatory mechanism of deriving eSCs from mES cells.

Conclusion: Conclusively, we have elucidated a differentiation roadmap of eSCs derived from mES cells with a relevant regulatory mechanism. Through co-overexpression of all these six factors, a large population of eSCs was successfully induced occupying 24% of the whole cell population (1 × 10⁵ cells/cm²). By adopting this approach, a mass of embryonic Sertoli cells can be generated for the purpose of co-culture technique, organ transplantation, gonadal developmental and sex determination researches.

Keywords: Embryonic Sertoli cells; Embryonic stem cells; Gonadogenesis; Lentiviral transduction; Male determinant factors; Molecular mechanism.

Fig. 1

Recovering mES cells from cryopreservation with MEF or TM4 feeder. a At day 3, PCs were able to aggregate in group mES+TM4. Scale bar = 400 μm. Enlarged figure shows on the right. b Growth curves of mES cells in mES+MEF, mES+TM4, mES+Trans, and mES+Trans+TM4 groups in the first 5 days. Results were expressed as mean ± SD (n = 3 independent experiments). Asterisks indicate statistical significance calculated by one-way ANOVA method and labeled between groups mES+TM4 and mES+MEF at days 4 and 5. c Five pluripotency markers of cells were detected in groups mES+MEF, mES+TM4, and mES+Trans by qPCR at day 5. qPCR results were expressed relative to the expression in group mES+MEF (control group). Results were expressed as mean ± SD (n = 3 independent experiments). Asterisks indicate statistical significance of differences in the mean gene expression according to the control group (mES+MEF). d Transcriptional expression of lentiviral transduced factors in mES cells were detected in groups mES+MEF, mES+Trans, and mES+Trans+TM4. qPCR results show changes in introduced factors among different groups in gene expression relative to expression of β-actin in self-comparison. Results were expressed as mean ± SD (n = 3 independent experiments). Asterisks indicate statistical significance of differences in the mean gene expression between the indicated groups. e Transcriptional level of some early gonadogenesis markers in mES+TM4 were expressed relative to the expression in group mES+MEF (control group) at day 5. Results were expressed as mean ± SD (n = 3 independent experiments). Asterisks indicate statistical significance of differences in the mean gene expression according to the control group (mES+MEF). (*P value < 0.05, **P value < 0.01, ***P value < 0.001)

Fig. 2

Development fate identification. a Stepwise development of progenitor cells to eSCs. b Heat map of stage-specific marker expression of different groups. In each transduction group, a different factor was removed from the pool of six factors. Group All factors was transduced with all the six factors. Group No factor was the control group (mES+MEF) without transfection. Group −Sry was the mES cells transduced with the six factors except for Sry. The rest were constructed in the same manner. Flow cytometry (FCM) with specific markers indicating different cell stages. AMH⁺/Emx2⁺ cells indicated coelomic epithelium. AMH⁺/SF1⁺ cells indicated SF1-positive precursor cells. AMH⁺/FasL⁺ cells indicated eSCs. Fluorescence antibody staining was performed after the samples had cell membrane perforation with Triton X-100. The results were expressed as mean value of the maximum positive cell portion of each test group in 35 days (n > 3 independent experiments). Gene expression levels of transcription factors c lin28, d FOG2, e dhh, and f Sox8 were detected by qPCR. Results show changes in gene expression relative to the highest expression in each group. c–f were expressed as mean ± SD (n = 3 independent experiments). Asterisks indicate statistical significance of differences in the mean of gene expression by one-way ANOVA according to the positive group (All factors) (*P value < 0.05, **P value < 0.01, ***P value < 0.001)

Fig. 3

Observing the induction process with FCM, IF, ICC, and qPCR. Optical micrographs a and b show the morphological characteristics of the PCs of mES+MEF and mES+Trans groups. In a, scale bar = 200 μm and 400 μm for the left and right panels, respectively. In b, scale bar = 200 μm. c Optical micrographs and IF were performed in group mES+Trans at day 20. Brightfield and fluorescence of AMH (green) images were merged on the left. Fluorescence of FasL (green) and Dapi (blue) merged imaging on the right. Scale bar = 200 μm. d ICC was performed with FasL antibody in groups mES+MEF and mES+Trans at day 30. Dark brown color shows the positive cells. Pale brown and achromatic colors show the negative cells. Scale bar = 400 μm. e At day 35 in group mES+Trans, tubular-like structure colonies were observed. Results show in optical micrographs on the left, and fluorescence merging images FasL (green) and Dapi (blue) on the right. Scale bar = 200 μm. f At day 30, FasL-positive cells formed ring-like structures in group mES+Trans. Scale bar = 100 μm

Fig. 4

Identification of morphological changes with biomarkers, external features, and FCM. The cell portion of FasL⁻/AMH⁺ and FasL⁺/AMH⁺ cells in group mES+Trans were detected via FCM. The result of the whole cell population of mES+Trans between 13 and 48 days is shown in a. The result of the cells removed of PCs between 13 and 34 days is shown in b. Tests were performed every 7 days and expressed as mean ± SD (n = 3 independent experiments). c Identification of the transcriptional level of epithelial markers and mesenchymal markers reveals the timing of morphological transformation (MET or EMT) between 5 and 45 days. qPCR was performed every 5 days and expressed relative to the highest expression among each marker. qPCR results took the mean value and are shown in the heat map (n = 3 independent experiments). MET mesenchymal to epithelial transformation. EMT epithelial to mesenchymal transformation. Heat map indicates the gene expression levels of major transcription factors in group mES+Trans for d pluripotency, onset of gonads, bi-potential gonads, and male gonads, and for e introduced factors. qPCR results show changes in gene expression relative to the highest expression in each marker. Results were expressed as mean and transformed into heat map (n > 2)

Fig. 5

Differentiation roadmap of generating Sertoli cells. A speculated stepwise derivation process of the mouse Sertoli cell including relevant molecular mechanisms. GATA4 and WT1 promote the generation of coelomic epithelium. SF1 increases the direct precursor cells of eSCs. Sry and Sox9 improve male determination. Dmrt1 maintains the male development of eSCs and suppresses female determination with Sox9