Candelaria Juliana I, Botigelli Ramon C, Guiltinan Carly, Shikanov Ariella, Denicol Anna C
bioRxiv. 2024 Aug 7:2024.07.18.604061. doi: 10.1101/2024.07.18.604061.
Here we explored poly(ethylene glycol) (PEG) bioengineered hydrogels for bovine preantral follicle culture with or without ovarian cell co-culture and examined the potential for differentiation of bovine embryonic stem cells (bESCs) towards gonadal somatic cells to develop a system more similar to the ovarian microenvironment.
Bovine preantral follicles were first cultured in two-dimensional (2D) control or within PEG hydrogels (3D) and then co-cultured within PEG hydrogels with bovine ovarian cells (BOCs) to determine growth and viability. Finally, we tested conditions to drive differentiation of bESCs towards the intermediate mesoderm and bipotential gonad fate.
Primary follicles grew over the 10-day culture period in PEG hydrogels compared to 2D control. Early secondary follicles maintained a similar diameter within the PEG while control follicles decreased in size. Follicles lost viability after co-encapsulation with BOCs; BOCs lost stromal cell signature over the culture period within hydrogels. Induction of bESCs towards gonadal somatic fate under WNT signaling was sufficient to upregulate intermediate mesoderm ( ) and early coelomic epithelium/bipotential gonad markers ( , , ). Higher BMP4 concentrations upregulated the lateral plate mesoderm marker . expression was not induced, indicating absence of the paraxial mesoderm lineage.
Culture of primary stage preantral follicles in PEG hydrogels promoted growth compared to controls; BOCs did not maintain identity in the PEG hydrogels. Collectively, we demonstrate that PEG hydrogels can be a potential culture system for early preantral follicles pending refinements, which could include addition of ESC-derived ovarian somatic cells using the protocol described here.
We demonstrate that three-dimensional bioengineered hydrogels could aid in the survival and growth of small bovine preantral follicles. Moreover, bovine embryonic stem cells have the potential to differentiate towards precursors of somatic gonadal cell types, presenting an alternative cell source for preantral follicle co-culture.
在此，我们探索了聚乙二醇（PEG）生物工程水凝胶用于牛腔前卵泡培养，有无卵巢细胞共培养的情况，并研究了牛胚胎干细胞（bESCs）向性腺体细胞分化以开发更类似于卵巢微环境系统的潜力。
首先将牛腔前卵泡在二维（2D）对照中或在PEG水凝胶（3D）中培养，然后在PEG水凝胶中与牛卵巢细胞（BOCs）共培养以确定生长和活力。最后，我们测试了促使bESCs向中间中胚层和双能性腺命运分化的条件。
与2D对照相比，初级卵泡在PEG水凝胶中10天的培养期内生长。早期次级卵泡在PEG中保持相似的直径，而对照卵泡尺寸减小。与BOCs共包封后卵泡失去活力；BOCs在水凝胶中的培养期内失去了基质细胞特征。在WNT信号传导下诱导bESCs向性腺体细胞命运分化足以上调中间中胚层（ ）和早期体腔上皮/双能性腺标记物（ 、 、 ）。较高浓度的BMP4上调了侧板中胚层标记物 。未诱导 表达，表明不存在轴旁中胚层谱系。
与对照相比，在PEG水凝胶中培养初级阶段的腔前卵泡可促进生长；BOCs在PEG水凝胶中不能维持其特性。总体而言，我们证明PEG水凝胶在有待改进的情况下可以成为早期腔前卵泡的潜在培养系统，改进措施可能包括使用本文所述方案添加ESC来源的卵巢体细胞。
我们证明三维生物工程水凝胶有助于小牛腔前小卵泡的存活和生长。此外，牛胚胎干细胞有潜力分化为性腺体细胞类型的前体，为腔前卵泡共培养提供了另一种细胞来源。
