Dataset Information

Continuous expression of reprogramming factors induces and maintains mouse pluripotency without specific growth factors and signaling inhibitors.

ABSTRACT:

Objectives

Derivation and maintenance of pluripotent stem cells (PSCs) generally require optimized and complex culture media, which hinders the derivation of PSCs from various species. Expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) can reprogram somatic cells into induced PSCs (iPSCs), even for species possessing no optimal culture condition. Herein, we explored whether expression of OSKM could induce and maintain pluripotency without PSC-specific growth factors and signaling inhibitors.

Methods

The culture medium of Tet-On-OSKM/Oct4-GFP mouse embryonic stem cells (ESCs) was switched from N2B27 with MEK inhibitor, GSK3β inhibitor, and leukemia inhibitory factor (LIF) (2iL) to N2B27 with doxycycline. Tet-On-OSKM mouse embryonic fibroblast (MEF) cells were reprogrammed in N2B27 with doxycycline. Cell proliferation was traced. Pluripotency was assessed by expression of ESC marker genes, teratoma, and chimera formation. RNA-Seq was conducted to analyze gene expression.

Results

Via continuous expression of OSKM, mouse ESCs (OSKM-ESCs) and the resulting iPSCs (OSKM-iPSCs) reprogrammed from MEF cells propagated stably, expressed pluripotency marker genes, and formed three germ layers in teratomas. Transcriptional landscapes of OSKM-iPSCs resembled those of ESCs cultured in 2iL and were more similar to those of ESCs cultured in serum/LIF. Furthermore, OSKM-iPSCs contributed to germline transmission.

Conclusions

Expression of OSKM could induce and maintain mouse pluripotency without specific culturing factors. Importantly, OSKM-iPSCs could produce gene-modified animals through germline transmission, with potential applications in other species.

SUBMITTER: Mao Y

PROVIDER: S-EPMC8349648 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Derivation and maintenance of pluripotent stem cells (PSCs) including embryonic stem cells(ESCs) and (iPSCs) usually requires optimization of complex culture media, which hinders the generation of PSCs from various species. Expression of Oct4, Sox2, Klf4 and c-Myc (OSKM) can reprogram somatic cells into iPSCs, even for species possessing no optimal culture condition. Here we explored whether expression of OSKM could induce and maintain pluripotency without specific growth factors and signaling inhibitors. Tet-On-OSKM/Oct4-GFP Mouse ESCs (mESCs) were derived from the embryos obtained after mating of Tet-On-OSKM mouse and Oct4-GFP mouse under the 2iL ( N2B27 plus MEK inhibitor PD0325901, GSK3β inhibitor ChIR99021, and leukemia inhibitory factor) condition. Tet-On-OSKM mouse was Gt(ROSA)26Sor tm1(rtTA*M2)Jae Col1a1 tm3(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae/J(The Jackson Laboratory,011004), and Oct4-GFP mouse was B6;CBA-Tg(Pou5f1-EGFP)2Mnn/J (The Jackson Laboratory,004654).The culture medium of Tet-On-OSKM/Oct4-GFP mouse ESCs was switched from 2iL to N2B27 plus Doxycycline medium (OSKM medium), under which OSKM trangene was activated. These above cells switched from 2iL to OSKM medium were called OSKM-ESCs. Meanwhile 2iL-ESCs (continuely cultured in 2iL) served as the positive control group. 2iL-ESCs and OSKM-ESCs collected at selective days (d18, 25 and 38) after medium switch were collected for RNA-Seq. Moreover, Tet-On-OSKM mouse embryonic fibroblast (MEF) cells were reprogrammed under OSKM medium (N2B27 plus Doxycycline) and 2iL medium, respectively. The resulting OSKM-iPSCs were reprogrammed and cultured under OSKM medium.The 2iL-iPSCs were reprogrammed under 2iL plus Doxycycline medium and cultured under 2iL medium (without Doxycycline)once clones were picked up.RNA-Seq of these above samples was conducted to analyze gene expression. We found that via continuous expression of OSKM,OSKM-ESCs and OSKM-iPSCs propagated stably, expressed pluripotency marker genes, and formed three germ layers in teratomas. Importantly, OSKM-iPSCs could produce gene-modified animals through germline transmission.Transcriptional landscapes of OSKM-iPSCs resembled 2iL-ESCs, and were more similar to those of ESCs cultured in serum/LIF.