MRPS18-2 protein immortalizes primary rat embryonic fibroblasts and endows them with stem cell-like properties

Abstract

We report that the overexpression of mitochondrial ribosomal protein MRPS18-2 (S18-2) can immortalize primary rat embryonic fibroblasts (REFs). The immortalized cells (18IM) lose contact inhibition, form foci, and are capable of anchorage-independent growth. Concurrently, mesodermal markers, such as vimentin, smooth muscle actin, and Fut4, disappear completely. 18IM cells express embryonic stem cell markers, such as SSEA-1, Sox2, and Oct3/4. In confluent cultures, a portion of cells also express ectoderm- and endoderm-specific pan-keratin, ectoderm-specific beta-III-tubulin, mesoderm-specific MHC class II, and become stainable for fat with Oil red O. None of these changes was detected in c-myc+Ha-ras (MR)-transformed cells. In immunodeficient mice, 18IM cells formed small transiently growing tumors that have down-regulated SSEA-1 and showed pan-keratin staining. We conclude that S18-2 can immortalize REFs and induces them to express stem cell traits.

Fig. 1.

Growth characteristic of S18–2 immortalized cells. (A) Colony formation assay. Primary rat embryonic fibroblasts were transfected with the different plasmids. After 2 weeks of selection on 0.5 mg/mL G418, the cells were fixed with formaldehyde and stained with crystal violet. (B) Fusion GFP-S18–2 protein expression (in green) in the 18IM cells. They were stained with mouse monoclonal anti-GFP antibody. DNA was stained with Hoechst (blue signal). Notice the mainly nuclear localization of GFP-S18–2 protein. (C) si-RNA treatment of the 18IM cells. Cells were treated with si-RNA for 48 h. Western blots were probed with rabbit polyclonal anti-S18–2 antibody for GFP-S18–2 fusion protein. (D) Growth of GFP-S18–2 transformed cells. Freshly passaged REFs did not form foci (first from the Left). The 18IM cells formed foci. Cells of different shapes were observed in the periphery (second from the Left). Notice increased number of mitoses in the 18IM cells. (E) Growth of GFP-S18–2 transformed cells. The 18IM cells formed embryoid body like structures in bacterial Petri dishes (Left). After 2–3 days these bodies fused with each other, forming large agglomerates after 3–5 days (Right). (F) Growth of c-myc and mutated Ha-ras transformed cells. REFs transformed by c-myc and mutated Ha-ras (MR cells) formed clumps in bacterial Petri dishes. (G) Growth of GFP-S18–2 transformed cells. The 18IM cells grew in soft agar (0.6% on the Bottom, 0,5% Top agarose) in contrast to primary REFs.

Fig. 2.

Comparative expression of differentiation markers by 18IM, MR cells, and REFs. (A) Immunostainings on primary rat embryonic fibroblasts (REFs) and GFP-S18–2 transformed cells (18IM). Green signal, marker expression. Blue signal, DNA (stained with Hoechst). (B) si-RNA treatment of GFP-S18–2 cells grown on cover slips. Top, untreated cells stained with anti-SSEA-1. Green, SSEA-1 signal; blue, DNA staining. Lower, cells treated with si-RNA for 24 h. Green, SSEA-1 signal; blue, DNA staining. SSEA-1 staining decreased significantly. (C) RT-PCR on primary rat fibroblasts (REFs) and GFP-S18–2 transformed cells (18IM). Marker (100-bp) was loaded in the first lane.

Fig. 3.

Expression of differentiation markers by 18IM cells. (A) Staining of the 18IM cells in confluent cultures. Suspended cells were collected and spun onto glass slides. Oil Red O staining. Red, fat cells. (B) Immunostaining of the suspended cells. Suspended 18IM cells were collected and spun onto glass slides. MHC class II staining. Green, MHC class II; blue, DNA. (C) Immunostaining of the 18IM cells in confluent cultures. The 18IM cells grown on coverslips were stained with antibody against beta III-tubulin. Green, beta III tubulin; blue, DNA. (D) Immunostaining of the 18IM cells. Pan-keratin is expressed in the differentiated from embryoid bodies 18IM cells, in contrast to REF and MR cells. (E) Immunostaining of cell agglomerates on frozen sections. Top, SSEA-1 expression. Notice decrease in the frequency of SSEA-1 positive cells (green) compared to the correspondingly cultured cells (Fig. 2 A and B, Top). DNA was stained with Hoechst (blue signal). Bottom, pan-keratin staining. Notice positive cells at the edges of the agglomerate (green signal). DNA is stained in blue.

Fig. 4.

Immunostaining of the tumors produced by GFP-S18–2 transformed cells in SCID mice. Formalin fixed paraffin embedded tissue was sectioned (5-μm). Paraffin was removed by xylene, followed by ethanol. Epitopes were recovered by boiling in microwave oven in citric buffer. Left: hematoxylin and eosin staining. Center: green, pan-keratin; blue, DNA. Heterochromatin in mouse cells forms aggregates in the nucleus, but is distributed evenly in rat cells. Only rat cells express pan-keratin. Right, DNA staining. Arrowheads, mouse cell nuclei; stars, rat nuclei.