Development of a Triple Genome-Edited Hypoimmunogenic Human-Induced Pluripotent Stem Cell Line which Allows In vivo Tracking of Cardiomyocyte Maturation upon Transplantation

 

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Background: Human induced pluripotent stem cells (hiPSCs) and their derivatives provide a promising cellular source for cardiac regenerative cell therapy. However, aspects such as immune rejection and evaluation of cell survival and maturation in vivo still need to be addressed. Therefore, we sought to develop a triple transgenic hiPSC line that is both hypoimmunogenic and allows tracking of cardiomyocyte maturation upon transplantation.

Method: We performed a total of three consecutive gene-editing steps in a human iPSC line using CRISPR/Cas9 technology. First, we introduced a td-Tomato reporter into the MLC2V gene, to detect mature ventricular cardiomyocytes (CMs) by red fluorescence. Second, we replaced the B2M gene, an important component of the MHC-I complex, with a neomycin cassette to reduce the response of allogeneic CD8+ cytotoxic T cells. Finally, we inserted a GFP reporter into the AAVS1 safe harbor locus to allow continuous detection of transplanted cells irrespective of their cellular status. The cell line was characterized after gene editing by qRT-PCR, FACS, immunocytochemistry (ICC), and Western blot. The immunological effects of the B2M knockout (KO) were evaluated in a lactate dehydrogenase (LDH) cytotoxicity assay.

Results: Successful gene editing was confirmed at all three steps by PCR and Sanger sequencing. The cell line remained fully pluripotent after three gene editing steps, demonstrated by TRA 1–60 and TRA 1–81 expression in FACS and ICC of NANOG and SOX. Expression of cardiac transcription factors and cardiac maturation markers by qRT-PCR and ICC of cardiac structural proteins during differentiation assays confirmed that cardiac development was not affected by gene editing. The B2M KO induced a significantly reduced immunologic response of CD8+ T-cells measured by LDH release. The reduction in the immunological response progressively increased during cardiac maturation. B2M KO CMs elicited a significantly reduced immune response compared with B2M wild-type CMs. GFP was continuously expressed at high level throughout cardiac differentiation at each time point, validated by qRT-PCR and FACS.

Conclusion: The generated triple transgenic hiPSC line combines hypoimmunogenic features with the ability to track transplanted cells in vivo at any stage of cardiac differentiation by two fluorescent reporters. It provides therefore a powerful cellular tool for the evaluation of different cell-based regenerative cardiac therapy approaches.

Publication History

Article published online:
03 February 2022

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