Efficient Selection of Knocked-In Pluripotent Stem Cells Using Dual Cassettes of Cellular Suicide System

Nakade, Koji; Tsukamoto, Sumihisa; Nakashima, Ken‐ichi; An, Yun‐Kyu; Sato, Iori; Li, Jingyue; Hemmi, Yasuko; Miwa, Yoshiro; Hayashi, Yohei

doi:10.2139/ssrn.4290057

Cited by 2 publications

(2 citation statements)

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“…To generate the EEF1A1-iRFP670 iPSC line, the expression vector for Cas9 nuclease and the guide RNA for cleaving the EEF1A1 gene, previously described as pX459-gEEF1A1 (RDB19570 deposited in the RIKEN DNA bank) (Nakade et al, 2023). The methods for constructing the donor vector for iRFP670 insertion were the same as those for PDX1-tdTomato, described previously.…”

Section: Methodsmentioning

confidence: 99%

“…The PDX1-tdTomato and EEF1A1-iRFP670 iPS lines were generated using genome editing technology modified from our method published in a previous study (Nakade et al, 2023). Briefly, the expression vector for Cas9 nuclease and the guide RNA for cleaving the PDX1 gene (RDB20280: pX330-PDX1) were constructed by inserting annealed synthetic oligomers (5′-CACCgcagctcctgcctctcatcg-3′ and 5′-AAACcgatgagaggcaggagctgc-3′) into the Bbs I site of pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (#42230; Addgene, Watertown, MA, USA; a gift from Prof. Feng Zhang).…”

Section: Generation Of Pdx1-tdtomato and Eef1a1-irfp670 Ips Lines Usi...mentioning

confidence: 99%

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Intraplacental injection of human iPSC-derived PDX1+ pancreatic progenitors prolongs Pdx1-deficient mice survival

Wakimoto,

Jeon,

Shahri

et al. 2024

Preprint

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Interspecies chimeras comprising human tissues have potential for use in disease modeling and regenerative medicine. Here, we successfully transplanted human induced pluripotent stem cell (iPSC)-derived PDX1+ pancreatic progenitor cells into Pdx1-deficient mouse embryos via intraplacental injection. The engrafted human cells predominantly localized to the duodenum, produced insulin, and extended the lifespan of Pdx1-/- mice by up to 10 days after birth. Transcriptomic analyses confirmed human pancreatic gene expression in human cells engrafted into the mouse duodenum. Our findings demonstrate the feasibility of generating interspecies chimeras with functional human pancreatic cells through in utero transplantation of lineage-committed progenitors. This approach circumvents developmental barriers while minimizing ethical concerns associated with PSCs. However, the incomplete rescue of the Pdx1-/- phenotype highlights the need for further research to enhance human cell engraftment and tissue integration. Overall, this study provides a foundation for developing human-animal chimera models for studying human development and regenerative therapies.

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Section: Methodsmentioning

confidence: 99%

Section: Generation Of Pdx1-tdtomato and Eef1a1-irfp670 Ips Lines Usi...mentioning

confidence: 99%