Peinkofer Gabriel, Maass Martina, Pfannkuche Kurt, Sachinidis Agapios, Baldus Stephan, Hescheler Jürgen, Saric Tomo, Halbach Marcel
Department of Internal Medicine III, University Hospital of Cologne, Cologne, Germany.
Center for Physiology and Pathophysiology, Institute of Neurophysiology, Medical Faculty, University of Cologne, Robert-Koch Str. 37, Cologne, 50931, Germany.
Stem Cell Res Ther. 2021 Jan 8;12(1):46. doi: 10.1186/s13287-020-02089-5.
Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo.
To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6-7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count.
The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM.
The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.
诱导多能干细胞衍生的心肌细胞（iPSC-CM）被视为心脏细胞替代治疗中很有前景的细胞类型，但尚不清楚发育阶段是否会影响它们在宿主组织中的存活及功能整合，而这对于长期治疗益处至关重要。为了研究这一点，我们首先在分化过程中的三个不同时间点测试了小鼠iPSC-CM在体外的细胞黏附能力，然后在体内检查了梗死心肌中细胞的存活情况以及电整合质量。
为了测试体外细胞黏附能力，将iPSC-CM接种在纤连蛋白包被的细胞培养皿和脱细胞心室细胞外基质（ECM）支架上。在固定时间段后，对稳定附着的细胞进行定量。对于体内实验，将表达增强型绿色荧光蛋白的小鼠iPSC-CM注射到成年小鼠的梗死心脏中。6至7天后，制备有活力的心室组织切片，以便记录移植的iPSC-CM和周围宿主心肌细胞的动作电位（AP）。之后，将切片裂解并制备基因组DNA，然后用于定量实时PCR以评估移植的iPSC-CM数量。
体外结果表明，第14天、第16天和第18天的iPSC-CM在细胞黏附能力上存在差异，第14天的iPSC-CM在ECM支架上附着的细胞数量最多。心肌内注射后，与第16天的iPSC-CM相比，第14天的iPSC-CM显示出显著更高的细胞数量。AP测量显示，电整合质量没有显著差异，第14天和第16天的iPSC-CM之间的AP特性只有微小差异。
本研究结果表明，移植时的发育阶段对于移植的iPSC-CM的存活至关重要。分化第14天的iPSC-CM在体内移植后显示出最高的存活率，这可能是由于其对细胞外基质的黏附能力更强。
