Abstract:
iPSC-derived human β-like cells (BLC) hold promise for both therapy and disease modelling, but their generation remains challenging and their functional analyses beyond transcriptomic and morphological assessments remain limited. Here, we validate an approach using multicellular and single cell electrophysiological tools to evaluate BLCs functions. The Multi-Electrode Arrays (MEAs) measuring the extracellular electrical activity revealed that BLCs are electrically coupled, produce slow potential (SP) signals like primary β-cells that are closely linked to insulin secretion. We also used high-resolution single-cell patch-clamp measurements to capture the exocytotic properties, and characterize voltage-gated sodium and calcium currents. These were comparable to those in primary β and EndoC-βH1 cells. The KATP channel conductance is greater than in human primary β cells which may account for the limited glucose responsiveness observed with MEA. We used MEAs to study the impact of the type 2 diabetes protective SLC30A8 allele (p.Lys34Serfs*50) and found that BLCs with this allele have stronger electrical coupling. Our data suggest that with an adapted approach BLCs from pioneer protocol can be used to evaluate the functional impact of genetic variants on β-cell function and coupling.
摘要:
iPSC衍生的人β样细胞(BLC)有望用于治疗和疾病建模,但是它们的产生仍然具有挑战性,并且它们在转录组学和形态学评估之外的功能分析仍然有限。这里,我们验证了使用多细胞和单细胞电生理工具评估BLC功能的方法。测量细胞外电活动的多电极阵列(MEAs)表明BLC是电耦合的,产生慢电位(SP)信号,如与胰岛素分泌密切相关的原代β细胞。我们还使用高分辨率单细胞膜片钳测量来捕获胞吐特性,并表征电压门控钠和钙电流。这些与原代β和EndoC-βH1细胞中的那些相当。KATP通道电导大于人原代β细胞,这可能是MEA观察到的有限葡萄糖响应性的原因。我们使用MEAs研究了2型糖尿病保护性SLC30A8等位基因的影响(p。Lys34Serfs*50),并发现具有该等位基因的BLC具有更强的电耦合。我们的数据表明,采用适应的方法,来自先驱方案的BLC可用于评估遗传变体对β细胞功能和偶联的功能影响。
■我们为什么进行这项研究?:来自开创性方案的iPSC衍生的β样细胞(BLC)以可变的β细胞功能和混合细胞群体而闻名,这极大地限制了下游功能评估。为了克服这一挑战,我们使用电生理工具对BLC进行了详细的功能评估.然后,我们希望应用这种方法来鉴定携带保护性2型糖尿病SLC30A8等位基因的BLC的其他功能差异。我们想要回答的具体问题是什么?:电生理学方法可以提供iPSC衍生的BLC的详细功能表征吗?这种方法是否足够敏感以捕获由SLC30A8功能丧失(lof)引起的功能差异?我们发现了什么?:我们发现从先驱方案产生的BLC与人类胰腺β细胞共享电生理特征,并且T2D保护性SLC30A8lof等位基因改善了人β细胞的电耦合活性。我们的发现有什么意义?:我们的发现验证了使用细胞内电生理学和细胞外电生理学来评估和监测BLC的功能。我们的方法打开了使用MEA实时监测iPSC衍生的BLC的分化质量并确定糖尿病相关变异的功能后果的观点。