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LIN28A inhibits DUSP family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells.

ABSTRACT: LIN28A, an RNA-binding protein, plays an important role in porcine induced pluripotent stem cells (piPSCs). However, the molecular mechanism underlying the function of LIN28A in the maintenance of pluripotency in piPSCs remains unclear. Here, we explored the function of LIN28A in piPSCs based on its overexpression and knockdown. We performed total RNA sequencing (RNA-seq) of piPSCs and detected the expression levels of relevant genes by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and immunofluorescence staining. Results indicated that piPSC proliferation ability decreased following LIN28A knockdown. Furthermore, when LIN28A expression in the shLIN28A2 group was lower (by 20%) than that in the negative control knockdown group ( shNC), the pluripotency of piPSCs disappeared and they differentiated into neuroectoderm cells. Results also showed that LIN28A overexpression inhibited the expression of DUSP (dual-specificity phosphatases) family phosphatases and activated the mitogen-activated protein kinase (MAPK) signaling pathway. Thus, LIN28A appears to activate the MAPK signaling pathway to maintain the pluripotency and proliferation ability of piPSCs. Our study provides a new resource for exploring the functions of LIN28A in piPSCs.

SUBMITTER: Wu XL

PROVIDER: S-EPMC8175949 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPluripotent stem cells (PSCs) offer immense potential as a source for regenerative therapies. The teratoma assay is widely used in the field of stem cells and regenerative medicine, but the cell composition of teratoma is still elusive.MethodsWe utilized PSCs expressing enhanced green fluorescent protein (EGFP) under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo. OCT4-MES (mouse embryonic stem cells) were isolated from the blastocysts of 3.5-day OCT4-EGFP mice (transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter) embryos, and TG iPS 1-7 (induced pluripotent stem cells) were generated from mouse embryonic fibroblasts (MEFs) from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. These pluripotent cells were characterized according to their morphology and expression of pluripotency markers. Their differentiation ability was studied with in vivo teratoma formation assays. Further differences between pluripotent cells were examined by real-time quantitative PCR (qPCR).ResultsThe results showed that several OCT4-expressing PSCs escaped differentiation inside of teratomas, and these escaped cells (MES-FT, GFP-positive cells separated from OCT4-MES-derived teratomas; and iPS-FT, GFP-positive cells obtained from teratomas formed by TG iPS 1-7) retained their pluripotency. Interestingly, a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT (MES-ST, GFP-positive cells isolated from MES-FT-derived teratomas; iPS-ST, GFP-positive cells obtained from teratomas formed by iPS-FT) were still pluripotent, as shown by alkaline phosphatase (AP) staining, immunofluorescent staining and PCR. MES-FT, iPS-FT, MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation, such as Dazl, Stella and Stra8.ConclusionsIn summary, a small number of PSCs escaped differentiation inside of teratomas, and these cells maintained pluripotency and partially developed towards germ cells. Both escaped PSCs and germ cells present a risk of tumor formation. Therefore, medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.

Project description:Understanding essential signaling network requirements and making appropriate adjustments in culture conditions are crucial if porcine pluripotent stem cells (PSC) are to achieve their full potential. Here, we first used two protein factors (LIF and FGF2) and kinase inhibitor combinations in attempts to convert primed type lentiviral-reprogrammed porcine induced PSC (Lv-piPSC) into naïve-like state and developed a medium called FL6i. In addition to FGF2 and LIF, this medium contained inhibitors of MAPK14, MAPK8, TGFB1, MAP2K1, GSK3A and BMP. Crucially, the usual TGFB1 and BMP4 protein components of many stem cell media were replaced in FL6i with inhibitors of TGFB1 and BMP. With this medium, Lv-piPSC were readily transformed from their original primed state into cells that formed colonies with typical features of naïve-state stem cells. The FL6i medium also assisted generation of naïve-type piPSC lines from porcine embryonic fibroblasts with non-integrating episomal plasmids (Epi-piPSC). These lines, despite retaining variable amounts of vector DNA, expressed higher endogenous pPOU5F1 and pSOX2 than Lv-piPSC. They have been cultured without obvious morphological change for >45 passages and retained pluripotent phenotypes in terms of upregulation of genes associated with pluripotency, low expression of genes linked to emergence of somatic cell lineages, and ability to generate well differentiated teratomas in immune-compromised mice. FL6i conditions, therefore, appear to support elevated pluripotent phenotypes. However, FL6i was less able to support the generation of embryonic stem cells from porcine blastocysts. Although colonies with dome-shaped morphologies were evident and the cells had some gene expression features linked to pluripotency, the phenotypes were ultimately not stable. Pathway analysis derived from RNAseq data performed on the various cell lines generated in this study suggest the benefits of employing the FL6i medium on porcine cells reside in its ability to minimize TGFB1 and BMP signaling, which would otherwise de-stabilize the stem cell state.

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LIN28A inhibits DUSP family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells.

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