Regulation of stem cell function and neuronal differentiation by HERV-K via mTOR pathway

Abstract

Stem cells are capable of unlimited proliferation but can be induced to form brain cells. Factors that specifically regulate human development are poorly understood. We found that human stem cells expressed high levels of the envelope protein of an endogenized human-specific retrovirus (HERV-K, HML-2) from loci in chromosomes 12 and 19. The envelope protein was expressed on the cell membrane of the stem cells and was critical in maintaining the stemness via interactions with CD98HC, leading to triggering of human-specific signaling pathways involving mammalian target of rapamycin (mTOR) and lysophosphatidylcholine acyltransferase (LPCAT1)-mediated epigenetic changes. Down-regulation or epigenetic silencing of HML-2 env resulted in dissociation of the stem cell colonies and enhanced differentiation along neuronal pathways. Thus HML-2 regulation is critical for human embryonic and neurodevelopment, while it's dysregulation may play a role in tumorigenesis and neurodegeneration.

Keywords: embryogenesis; endogenous retrovirus; neurogenesis; progenitor cells; stem cells.

Fig. 1.

Activation of HML-2 Env in PSC and silencing upon differentiation. (A) CD34⁺ hematopoietic stem cells were isolated from blood samples and infected with Sendai virus particles containing Yamanaka factors Oct-4, C-Myc, Klf-4, and Sox-2 to generate iPSC. Five days later, HML-2 env expression in the infected cells and control CD34 cells was monitored by qPCR. Data represent mean ± SEM from four independent experiments. (B) iPSC were differentiated into NSC using neural induction medium, HML-2 env expression in NSC was significantly decreased compared with iPSC. Data represent mean ± SEM from four independent experiments. (C) Similarly, ES cell line W9 had high levels of HML-2 env expression but declined upon differentiation to NSC. Data represent mean ± SEM from five independent experiments. (D) Western blot analysis showed that expression of HML-2 Env in the iPSC but no expression was seen at the NSC or neurons. Data are mean ± SEM from four independent experiments. (E) Immunostaining for HML-2 Env (green) shows HML-2 Env expression in iPSC (Env: green; stem cell marker Oct3/4: red; DAPI: blue). Antibody control (Ab ctrl) represents immunostaining without primary antibodies. Images are representative of three independent experiments. (Scale bar, 200 μm.) (F) Immunostaining of the iPSCs with an antibody of HML-2 Env (green) shows prominent immunostaining in the iPSC membrane, which decreased when the cells were treated with detergents, saponin, or triton after fixation with PFA. (Scale bar, 100 µm.) Images are representative of three independent experiments. (G) iPSC were immunostained for HML-2 Env (red) and TRA1-60 (green) directed against the iPSC membrane. (Scale bar, 20 µm.) DAPI (blue) was used to stain the nuclei. (H) Cell membrane staining of HML-2 Env and TRA1-60 on isolated live iPSCs was determined by flow cytometry. (i) No staining was observed in cells incubated with only secondary antibodies conjugated with fluorophores, (ii) Cells immunostained with antibodies against HML-2 and TRA1-16, showed that 92.3% of the cells were TRA1-60⁺, and 89% were also HML-2 Env⁺.

Fig. 2.

Determination the loci of HML-2 env expression. One-kilobase transcripts from the HML-2 env derived from iPSCs were amplified, cloned, and sequenced and 14 sites were found transcriptionally active. (A) Diagram shows where the HML-2 Env coding sequences and the PCR primers are. (B) Schematic of HML-2 env genes in the human genome expressed in iPSC are shown. Solid lines indicate the length of the transcript. The 12q14.1 env has a full-length transcript except for a 3-nt deletion. The 19q11 env has no deletion and no premature stop codon. (C) RNA-seq data from three iPSC lines—PAU, NC4 and WTC11—were used with “samtools” and the available command supported by that tool called “bedcov” to generate the coverage values for the two env loci. It showed that 19q11 predominantly produced the env reads.

Fig. 3.

Inhibition of HML-2 env decreases stemness and stem cell colony formation. (A) Specific Ei and Gi were used to treat the iPSC. Oct-4 transcripts were decreased by Ei treatment in iPSC compared to control Nsi after 24 h. Data represents mean ± SEM from three independent experiments. (B) The effect on cell viability was monitored using cellquanti blue assay. No significant changes were observed in cell numbers following treatment with Ei or Gi compared to Nsi. Data represent mean ± SEM from three independent experiments. (C) After 48 h of treatment, cells were fixed and stained for HML-2 Env (green) and DAPI (blue) and imaged by a TIRF microscope. Decrease in HML-2 expression and an increase in cell size was observed in the cells treated with Ei compared with Nsi, while no significant effect of Gi treatment was observed. Data represent mean ± SEM from three independent experiments. (Scale bar, 10 µm.) (D) iPSCs were dissociated with ETDA treatment and replated on Matrigel-coated plates. After 48 h, iPSC colonies were observed which immunostained for HML-2 Env (red) and stem cell marker Oct4 (green). (Scale bars, 100 µm.) (E) iPSCs were similarly dissociated and cultured in the presence or absence of control IgG or monoclonal antibody to HML-2 Env (mAb Env), or (F) treated with Nsi or Ei. The number of stem cell colonies were counted and expressed as a percentage of control. Data represents mean ± SEM from three independent experiments.

Fig. 4.

Inhibition of HML-2 Env promotes neuronal differentiation of stem cells. (A) Three days after iPSCs were transfected with Ei, and incubated in neural induction media, expression of NSC marker nestin was increased compared to control cells (Ctrl) and cells transfected with Nsi, as determined by immunostaining (blue: DAPI; green: nestin). (Scale bar, 50 µm.) (B) Corresponding Western blot analysis shows nestin in the Ei-treated cell extracts. Data are presented as mean ± SEM from three independent experiments. (C) After 7 d of further differentiation in neuronal medium, the cells assumed a neuronal morphology with immunostaining for β-III-tubulin (green). (Scale bars, 50 µm.) (D) iPSCs were treated with control IgG or antibody to HML-2 Env (Anti-Env). Increased immunostaining for nestin (green) was noted with anti-Env. (Scale bar, 50 µm.) (E) Corresponding Western blot analysis shows increased nestin with anti-Env treatment but not with control IgG or antibody to the transmembrane domain of the HML-2 Env (anti-TM). (F) Forced expression of HML-2 Env during iPSC neuronal differentiation by transfecting iPSC with a CMV promoter-driven Env plasmid resulted in delayed neuronal differentiation as shown by lack of 3D structure build-ups and (G) decreased expression of nestin expression by immunostaining (green for nestin, blue for nuclei DAPI staining), and (H) Western blot analysis. β-Actin was used as a loading control. (Scale bars, 400 µm in F and 25 µm in G.) Images are representative of three independent experiments.

Fig. 5.

Interaction between HML-2 Env and CD98HC. (A) iPSC lysates were immunoprecipitated using an antibody to HML-2 Env (anti-Env) and analyzed by Western blot using an antibody to CD98HC. Images are representative of three independent experiments (B) Immunostaining of stem cells showed colocalization of CD98HC (red) and HML-2 Env (green). (Scale bar, 10 µm.) (C) Forty-eight hours after transfection with Ei, CD98HC expression was decreased compared with Nsi. Data are presented as mean ± SEM from three independent experiments. (D) iPSCs were transfected with either Nsi or siRNA to HML-2 env. Inhibition of Env with Ei resulted in decreased expression of HML-2 Env (green) and CD98HC (red), as shown by immunostaining. (Scale bar, 20 µm.) (E) iPSCs were treated with either Nsi or siRNA to CD98HC (CD98HC si). Treatment with CD98HC si resulted in an increase in nestin⁺ cells (red) during neural induction as shown by immunostaining. (Scale bar, 100 µm.) (B, D, and E) DAPI (blue) was used to stain the nuclei. Images are representative of three independent experiments.

Fig. 6.

HML-2 Env regulates gene expression in iPSCs. HML-2 Env expression was inhibited by treating iPSCs with siRNA targeting HML-2 env in three different lines. Total RNA was collected for RNA-seq analysis. (A) Vertical bar plot describes genes dysregulated between Nsi and Ei (uncorrected ANCOVA P < 0.05, linear fold-change ≥ 1.5×). Genes are ranked by linear fold-change (blue bars = down-regulated, red bars = up-regulated). (B) These genes were imported into the IPA tool (https://www.qiagen.com/us/) to identify the top enriched functions by Exact Test and (C) the top enriched pathways by Exact Test. Number of down-regulated genes and up-regulated genes overlapping per function and pathway are described (x axis) along with the corresponding enrichment P value (y axis). (D) The top-scoring molecular network.

Fig. 7.

Interactions among HML-2 Env, CD98HC, and LPCAT1. (A) iPSCs immunostained for CD98HC (red) and LPCAT1 (green) showed LPCAT1 expression in CD98HC⁺ cells. DAPI (blue) was used to stain the nuclei. (Scale bar, 20 μm.) (B) iPSC were treated with either Nsi or siRNA to CD98HC for 24 h. LPCAT1 expression was decreased by siRNA targeting CD98HC as determined by Western blot analysis. (C) iPSC were also treated with either Nsi or Ei. Inhibition of env with Ei resulted in decreased LPCAT1 expression, as detected by RT-PCR and (D) Western blot analysis. (E) Inhibition of HML-2 env or CD98HC using siRNA resulted in a decrease in rpS6 production as determined by a Western blot analysis. (F) Effect of rapamycin (Rap) on the LPCAT1 production in iPSC was determined using Western blot analysis. (G) iPSCs were treated with siRNA to LPCAT1 for 24 h which resulted in increased nestin transcripts and 72 h after neuronal induction, an increase in nestin protein was noted (H), as detected by Western blot assay. Data are presented as mean ± SEM and images are representative of three independent experiments.

Fig. 8.

Introduction of HML-2 Env expression in rhesus NSC activates mTOR and LPCAT1 pathway. (A) Induced rhesus NSC positive-stained with nestin (green) could be differentiated to β-III-tubulin⁺ (red) neurons. (B) The rhesus NSCs were transfected with HML-2 Env plasmid for 48 h, which resulted in the increased levels of rpS6 and LPCAT1, as determined by Western blot assay. β-Actin or β-tubulin was used as a loading control. Images are representative of three independent experiments.