Radojevic Bojana, Conley Shannon M, Bennett Lea D
Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73114, USA.
Department of Cell Biology, Oklahoma Center for Geroscience and Healthy Brain Aging, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
J Dev Biol. 2021 Sep 10;9(3):38. doi: 10.3390/jdb9030038.
Human induced pluripotent stem cells (iPSCs) are differentiated into three-dimensional (3D) retinal organoids to study retinogenesis and diseases that would otherwise be impossible. The complexity and low yield in current protocols remain a technical challenge, particularly for inexperienced personnel. Differentiation protocols require labor-intensive and time-consuming dissection of optic vesicles (OVs). Here we compare this method with a suspension method of developing retinal organoids. iPSCs were differentiated with standard protocols but the suspension-grown method omitted the re-plating of embryoid bodies and dissection of OVs. All other media and treatments were identical between developmental methods. Developmental maturation was evaluated with RT-qPCR and immunocytochemistry. Dissection- and suspension-derived retinal organoids displayed temporal biogenesis of retinal cell types. Differences in retinal organoids generated by the two methods of differentiation included temporal developmental and the organization of neural retina layers. Retinal organoids grown in suspension showed delayed development and disorganized retinal layers compared to the dissected retinal organoids. We found that omitting the re-plating of EBs to form OVs resulted in numerous OVs that were easy to identify and matured along a retinal lineage. While more efficient, the suspension method led to retinal organoids with disorganized retinal layers compared to those obtained using conventional dissection protocols.
人类诱导多能干细胞（iPSCs）被分化为三维（3D）视网膜类器官，以研究视网膜发生过程以及那些用其他方法无法研究的疾病。当前方案的复杂性和低产量仍然是一项技术挑战，尤其是对于缺乏经验的人员而言。分化方案需要对视泡（OVs）进行劳动强度大且耗时的解剖。在这里，我们将这种方法与一种培养视网膜类器官的悬浮方法进行比较。iPSCs采用标准方案进行分化，但悬浮培养方法省略了胚状体的重新接种和视泡的解剖。两种发育方法之间的所有其他培养基和处理均相同。通过逆转录定量聚合酶链反应（RT-qPCR）和免疫细胞化学评估发育成熟度。解剖法和悬浮法衍生的视网膜类器官均显示出视网膜细胞类型的时序性发生。两种分化方法产生的视网膜类器官的差异包括发育时序以及神经视网膜层的组织结构。与解剖得到的视网膜类器官相比，悬浮培养的视网膜类器官发育延迟且视网膜层紊乱。我们发现，省略胚状体重新接种以形成视泡会产生大量易于识别且沿视网膜谱系成熟的视泡。虽然悬浮法更高效，但与使用传统解剖方案获得的视网膜类器官相比，它导致视网膜类器官的视网膜层紊乱。
