Linc-NSC affects cell differentiation, apoptosis and proliferation in mouse neural stem cells and embryonic stem cells in vitro and in vivo

Abstract

Background: Stem cell therapy is a promising therapeutic strategy. In a previous study, we evaluated tumorigenicity by the stereotactic transplantation of neural stem cells (NSCs) and embryonic stem cells (ESCs) from experimental mice. Twenty-eight days later, there was no evidence of tumor formation or long-term engraftment in the NSCs transplantation group. In contrast, the transplantation of ESCs caused tumor formation; this was due to their high proliferative capacity. Based on transcriptome sequencing, we found that a long intergenic non-coding RNA (named linc-NSC) with unknown structure and function was expressed at 1100-fold higher levels in NSCs than in ESCs. This finding suggested that linc-NSC is negatively correlated with stem cell pluripotency and tumor development, but positively correlated with neurogenesis. In the present study, we investigated the specific role of linc-NSC in NSCs/ESCs in tumor formation and neurogenesis.

Methods: Whole transcriptome profiling by RNA sequencing and bioinformatics was used to predict lncRNAs that are widely associated with enhanced tumorigenicity. The expression of linc-NSC was assessed by quantitative real-time PCR. We also performed a number of in vitro methods, including cell proliferation assays, differentiation assays, immunofluorescence assays, flow cytometry, along with in vivo survival and immunofluorescence assays to investigate the impacts of linc-NSC on tumor formation and neurogenesis in NSCs and ESCs.

Results: Following the knockdown of linc-NSC in NSCs, NSCs cultured in vitro and those transplanted into the cortex of mice showed stronger survival ability (P < 0.0001), enhanced proliferation(P < 0.001), and reduced apoptosis (P < 0.05); the opposite results were observed when linc-NSC was overexpressed in ESCs. Furthermore, the overexpression of linc-NSC in ECSs induced enhanced apoptosis (P < 0.001) and differentiation (P < 0.01), inhibited tumorigenesis (P < 0.05) in vivo, and led to a reduction in tumor weight (P < 0.0001).

Conclusions: Our analyses demonstrated that linc-NSC, a promising gene-edited target, may promote the differentiation of mouse NSCs and inhibit tumorigenesis in mouse ESCs. The knockdown of linc-NSC inhibited the apoptosis in NSCs both in vitro and in vivo, and prevented tumor formation, revealing a new dimension into the effect of lncRNA on low survival NSCs and providing a prospective gene manipulation target prior to transplantation. In parallel, the overexpression of linc-NSC induced apoptosis in ESCs both in vitro and in vivo and attenuated the tumorigenicity of ESCs in vivo, but did not completely prevent tumor formation.

Keywords: Genetic manipulation; Stem cells; Transplantation; Tumorigenicity.

Fig. 1

Prediction of candidate genes in NSC/ESC. A Heatmap map of predicted new lncRNAs in NSC/iNSC and ESC/iPSC. B Linc-NSC differential expression. C and D GO analysis (E) KEGG analysis. E The structures shown were predicted by Mfold

Fig. 2

Identification of the NSCs and modified NSCs. A Using Nestin (green) as a marker we identified NSC in 2D adherent culture. B After 72 h, fluorescence microscopy was used to detect transfection efficiency, and the transfection efficiency was > 60%. The infected cells were then selected in medium with 1ug/ml of puromycin for 3 days. Scale bar = 100 µm C Linc-NSC expression was measured by qPCR to assess the knockdown efficiency of linc-NSC (n = 3, one-way ANOVA with post hoc test, ***P < 0.001)

Fig. 3

Linc-NSC knockdown promotes the proliferation and inhibits the differentiation and apoptosis of NSCs in vitro. A Cell doubling time studies in shRNA group and shRNA control group cells. The shRNA cells had a population doubling time of less 24 h and the shRNA control cells of about 72 h. B Stemness and neurogenic properties was evaluated by the expressions of three pluripotency genes (Sox2, Oct4, and Nanog) and two neurogenic related gene (Map2 and S100b) after knockdown linc-NSC. An asterisk indicates that the difference between controls and Sox2 is significant. C and D Flow cytometric analysis of single-cell suspension prepared from Accutase-digested sphere. The apoptosis rate was defined as the early apoptosis rate plus the late apoptosis rate. E and F Image of the neurosphere in adherent culture immunostained with Nestin and differentiation marker (GFAP, NeuN and Olig2). Green color, Nestin staining; purple, GFAP staining; red, NeuN staining; orange, Olig2 staining; blue, DAPI. Scale bar = 20 µm. (n = 5, Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 4

Linc-NSC inhibited the proliferation and promoted apoptosis of ESCs in vitro. A The normal embryonic stem cells (ESC) were subjected to stable transfection with green fluorescent protein (GFP). Scale bar = 50 µm. B The ESCs were sub-cultured on mouse embryonic fibroblasts (MEFs) as feeder cells in embryonic stem cell medium. Scale bar = 100 µm. C The transfection efficiency of linc-NSC was assessed by measuring Linc-NSC expression using quantitative polymerase chain reaction (qPCR). (n = 3 qPCR replicates). D Cell doubling time studies were conducted on the OE group and OE control group cells, with cellular viability analyzed using the CCK8 assay (n = 5 duplicates). The OE group cells exhibited a population doubling time of less than 40 h, while the OE control cells had a population doubling time of approximately 36 h. E Ki67 expression in the OE group and OE control group was evaluated through immunofluorescence staining. Scale bar = 100 µm. F The cell cycle was analyzed through flow cytometry, with statistical results presented. (n = 3, Student’s t-test, *p < 0.05, ***p < 0.001, ****p < 0.0001). G Apoptosis detection was conducted through flow cytometry, with statistical results reported (n = 3, Student’s t-test, ***p < 0.001)

Fig. 5

Linc-NSC inhibited the stemness maintenance of ESCs in vitro. A Immunofluorescence staining for the stem cell pluripotency markers Sox2 (red), Nanog (red) and Oct4 (red). Scale bar = 100 µm. B The statistical analysis of immunofluorescence (n = 3, Student’s t-test, *p < 0.05)

Fig. 6

Linc-NSC promoted the neurogenic differetiation capabilities of ESCs in vitro. A Immunofluorescence staining for the stem cell differentiation markers NeuN (red), GFAP (red), and Olig2 (red). Scale bar = 100 µm. B The statistical analysis of immunofluorescence (n = 3, Student’s t-test,*p < 0.05, **p < 0.01)

Fig. 7

Linc-NSC enhanced the survival effect of NSC transplantation under the premise of non‐tumorigenicity. A A schematic depiction of the experiment conducted is presented. B The transplanted mouse brain's gross appearance is illustrated, and no macroscopic gross tumor formation was observed across all groups. C The explanted grafts were subjected to HE staining at two weeks (× 10, × 200) and four weeks (× 10, × 200) post-implantation. D The presence of GFP-labeled cells was detected through green fluorescent imaging of consecutive sections, with the injection needle tract indicated by an arrow. Scale bar = 100 µm. E The number of surviving grafted cells was quantified (n = 5, Student’s t-test, ****p < 0.0001, no statistically significant difference denoted by "ns")

Fig. 8

Linc-NSC suppressed the stemness properties of NSCs. A High power magnification of grafts two weeks and four weeks post-transplantation revealed immunofluorescence staining of GFAP (purple), NeuN (red), and Olig2 (orange). Scale bar = 20 µm. B The rates of GFAP + , NeuN + , and Olig2 + cells were subjected to statistical analysis between the shRNA and shRNA control groups (n = 5, Student’s t-test, **p < 0.01, no statistically significant difference denoted by "ns")

Fig. 9

Linc-NSC mitigated the tumorigenicity of ESCs transplantation by promoting differentiation. A and B Tumors were collected from the sacrificed mice, and the size of tumors was compared. C HE staining for the tumor formed 2 and 4 weeks (× 10, × 200) after cell transplantation. Scale bars = 1000 μm (left) and 50 μm (right). D Effect of linc-NSC on neurogenesis (GFAP + , NeuN + , and Olig2) of tumor tissue 28 days after cell transplantation. Scale bars = 100 μm. E Western blot assay of GFAP and NeuN in brain tumor tissue formed 28 days after cell transplantation. F Statistical graph of relative protein expression. (n = 3, Student’s t-test, *p < 0.05, **p < 0.01, no statistically significant difference denoted by "ns"). G TUNEL staining. Scale bar = 20 μm; DAPI: blue, TUNEL: green. H Quantitative analysis of TUNEL-positive cells (n = 5, one-way ANOVA, ****p < 0.0001)