CRISPR-Cas9/HDR介导Tet-On-MBX系统定点整合构建DOX可控永生化巨核祖细胞潜能的hiPSC系OA

Objective Overexpression of c-MYC,BMI1 and BCL-XL(collectively referred to as MBX)can confer immortalization potential on human induced pluripotent stem cell(hiPSC)-derived megakaryocyte progenitors,which may serve as seed cells for in vitro platelet production.However,existing construction strategies are limited by the risks of random integration and basal leaky expression.Using CRISPR-Cas9-mediated homology-directed repair(HDR),this study was designed to integrate the trans-regulatory element M2rtTA of the Tet-On system and the MBX sequence driven by the cis-acting response element TRE into the safe harbor loci ROSA26 and AAVS1 of the hiPSC genome,respectively,aiming to construct a doxycycline(DOX)-inducible hiPSC line with the potential to generate immortalized megakaryocyte progenitors.Methods ① Recombinant single guide RNA plasmids targeting the AAVS1 and ROSA26 loci,namely px458-sgAAVS1 and px458-sgROSA26,as well as the donor plasmids pAAVS1-TRE-MBX-EGFP and pROSA26-Hygro-M2rtTA were constructed.② The CAG-driven trans-regulatory element M2rtTA was integrated into the ROSA26 locus to generate CAG-M2rtTA hiPSCs.③ Based on the constructed CAG-M2rtTA hiPSCs,the TRE-driven MBX tandem cassette and EGFP reporter gene were integrated into the AAVS1 locus to generate CAG-M2rtTA/TRE-MBX hiPSCs.④ The cells were divided into an induction group(DOX-ON),a non-induction group(non-DOX)and a wild-type blank control group(WT).Differences in EGFP and MBX expression after DOX induction were detected by fluorescence microscopy and qPCR.⑤The 3 groups of cells were subjected to synchronous directed megakaryocytic differentiation.For the DOX-ON group,2 μg/mL DOX was added to the culture medium on day 12 of differentiation,and this culture condition was maintained until day 30.During this period,the cells were passaged normally,and EGFP expression was observed.On day 18 of differentiation,cell samples were harvested from the 3 groups.Wright-Giemsa staining and flow cytometry(detection of CD41a,CD42b and DNA ploidy)were performed to evaluate the regulatory effect of DOX on the directed differentiation of CAG-M2rtTA/TRE-MBX hiPSC cells into mature megakaryocytes.Meanwhile,part of the DOX-ON cells collected on day 18 were subjected to DOX withdrawal to establish the DOX-OFF group.Following drug withdrawal,these cells were cultured for another 6 d under the same differentiation conditions as the other groups,after which the expression of CD41a and CD42b was detected by flow cytometry.The experiment was performed with 3 biological replicates,and statistical analysis was conducted using the independent-samples t test.Results ①Sanger sequencing confirmed the successful construction of the targeting plasmids px458-sgAAVS1 and px458-sgROSA26.PCR analysis showed that M2rtTA and MBX-EGFP were precisely integrated into the ROSA26 and AAVS1 loci,respectively,yielding CAG-M2rtTA/TRE-MBX hiPSCs.② After 48 h of DOX induction,EGFP fluorescence was observed in the DOX-ON group.qPCR showed that the mRNA expression levels of c-MYC,BMI1 and BCL-XL were(14.78±1.28)-fold(P＜0.000 1),(7.87±0.24)-fold(P＜0.000 1)and(6.70±0.11)-fold(P=0.001 0)-fold higher than those in the WT group,respectively.No marked difference was observed between the non-DOX and WT groups.③ During the megakaryocytic differentiation,the DOX-ON group maintained sustained EGFP expression and proliferative capacity until day 30 of differentiation and passage 5.On day 18 of differentiation,the DOX-ON group retained megakaryocyte progenitor-like morphology,with a DNA ploidy＞4N cell proportion of 5.0％,and CD41a+CD42b-and CD41a+CD42b+cell proportions of 48.1％and 10.9％,respectively.The non-DOX group exhibited mature megakaryocyte-like morphology,with a DNA ploidy＞4N cell proportion of 17.9％,and CD41a+CD42b-and CD41a+CD42b+cell proportions of 22.3％and 40.7％,respectively,showing results consistent with that of the WT group.④ At 6 d after DOX withdrawal,the proportion of CD41a-CD42b-cells in the DOX-OFF group decreased to 19.9％,whereas that of CD41a+CD42b+cells increased to 33.5％,suggesting that the cells restored their terminal megakaryocytic differentiation capacity after MBX expression was switched off.Conclusion The CAG-M2rtTA/TRE-MBX hiPSC line has been successfully constructed via CRISPR-Cas9-mediated HDR.This cell line can maintain stable proliferation at the megakaryocyte progenitor stage through DOX-induced MBX overexpression.