Immortalization of primary endothelial cells (HUVEC) using viral gene transfer and

Recombinant Gfp-encoding LV particles could be shown to infect standard cell lines efficiently (see Fig. 3.5). These LV particles were produced using the same plasmid background as the oncogene-carrying LV particles needed for immortalization. The next step was to test the infection efficiency in primary cells.

Primary HUVEC (human umbilical vein endothelial cells) were infected with Gfp-encoding LV particles and the infection rate was quantified. For this purpose, samples were analyzed for the Gfp-positive population 5 days p.i. (Fig. 3.7). The primary HUVEC were infected with MOI 1.6. An infection rate of approximately 61 % was observed. This experiment showed efficient infection of primary cells by the recombinant Gfp-encoding LV particles. The result is comparable to the infection rate in Hela229 cells (see Fig. 3.5) with 50 % infected cells using MOI 1.

Results

Fig. 3.7: Recombinant Gfp-encoding LV particles infect primary HUVEC efficiently.

Quantification of infection rate by FACS analysis. Primary HUVEC infected with Gfp-encoding LV particles were analyzed for the Gfp-positive population in comparison to non-infected cells 5 days p.i.. An infection rate of approx. 61 % was observed in primary HUVEC infected with MOI 1.6. The histogram displays cell count against fluorescence intensity. The red bar designates the Gfp-positive cell population.

The next aim was to generate immortalized HUVEC by using the oncoproteins hTERT, SV40T and Bmi1 to analyze the effects of these oncoproteins on primary cells. All three oncoproteins were used individually as well as in combination of two each. Primary HUVEC were infected in parallel with LV particles carrying the different oncogenes with approximately MOI 1 and grown continuously. Four months after infection with LV particles, pictures were taken to visualise different growth characteristics. Cells expressing hTERT + SV40T were rounder and more regular shaped compared to all other samples (Fig. 3.8). Among the cells transduced with one of the oncogenes individually, only SV40T expressing cells continued proliferating beyond passage 29.

Thus, hTERT or Bmi1 alone were not sufficient for immortalization. However, comparing SV40T expressing cells with double-transduced cells could indicate incomplete immortalization, as SV40T expressing cells grew less steadily and more cells were enlarged or died. Four samples of transduced cells survived: HUVEC immortalized with hTERT + SV40T, hTERT + Bmi1, SV40T + Bmi1 and SV40T alone. The cells were cultivated up to p. 50 before original continuous culture was stopped.

Results

Fig. 3.8: Differences in cell viability and phenotype of immortalized HUVEC due to combination of oncogenes. Phase contrast pictures of transduced HUVEC four months post transduction. Primary HUVEC cells were infected with LV particles carrying different oncogenes as indicated and grown in parallel continuously. Pictures were acquired on the same day and passage numbers (p.) are indicated to visualise different growth characteristics. A) HUVEC immortalized with hTERT + SV40T grew steadily and had a round phenotype. B) HUVEC with hTERT + Bmi1 grew continuously, they exhibited longer and more irregular cell shapes. C) HUVEC with SV40T + Bmi1 grew steadily. Many cells appeared round with long cell shapes in between. D) HUVEC with hTERT alone had lowest survival rates. Cells had stopped growing in p. 19 and died completely after additional step of subculture. E) HUVEC with SV40T alone grew continuously. Cells showed mixed phenotypes with long as well as irregular cell shapes. F) HUVEC with Bmi1 alone had diminished survival rates. Enlarged irregular cells were observed, which died after additional step of subculture.

The successfully growing transduced HUVEC were considered to be immortalized. Next, gene expression of the oncogenes was verified in HUVEC immortalized with two of the oncogenes. The oncogene expression according to the inserted constructs could be verified in all three HUVEC samples transduced with two oncogenes (Fig. 3.9). Primary HUVEC did not express HTERT, SV40T or BMI1. HTERT was expressed in all HUVEC transduced with the hTERT construct (Fig. 3.9A). HUVEC immortalized with hTERT + SV40T showed 1.6-fold higher HTERT expression level than HUVEC immortalized with hTERT + Bmi1. All HUVEC carrying the SV40T construct showed expression of SV40T (Fig. 3.9B). HUVEC immortalized with hTERT + SV40T showed a 3.6-fold lower expression level than HUVEC immortalized with SV40T plus Bmi1. Both HUVEC immortalized with Bmi1 construct expressed BMI1 (Fig. 3.9C). However, BMI1 expression was 16.5-fold lower in HUVEC immortalized with hTERT + Bmi1 compared to HUVEC immortalized with SV40T + Bmi1.

Notably, HUVEC immortalized with SV40T + Bmi1 showed highest expression of both transduced oncogenes SV40T and BMI1 among all HUVEC. TMNK-1 cells served as

Results

Fig. 3.9: Verification of oncogene expression in immortalized HUVEC by real time RT-PCR.

Quantitative real time RT-PCR was performed to determine the expression of oncogenes on mRNA level in immortalized HUVEC cells compared to primary HUVEC cells, TMNK-1 cells and Hela229 cells. Relative expression values were normalized to primary HUVEC (sample 1). hT:

hTERT; SV: SV40T; Bmi: Bmi1. A) Relative expression of HTERT. HTERT was expressed in all HUVEC transduced with the hTERT construct (samples 2, 3) and in TMNK-1 cells. B) Relative expression of SV40T. All HUVEC carrying the SV40T construct (samples 2, 4) and TMNK-1 cells showed expression of SV40T. C) Relative expression of BMI1. HUVEC carrying Bmi1 construct

Results

positive control and expressed HTERT as well as SV40T as expected (see above).

Hela229 cancer cells expressed HTERT at very low levels (64-fold lower compared to TMNK-1 HTERT expression). As oncogene expression was verified for the respective genes, the immortalization of primary HUVEC was successful.

After immortalization of primary cells, characterization of obtained cells was necessary to verify endothelial origin and to exclude changes of endothelial phenotype. Therefore, the four surviving immortalized HUVEC samples were analyzed for expression of the endothelial marker platelet/endothelial cell adhesion molecule (PECAM-1) (Fig. 3.10). All cell samples were positive for PECAM-1, which localized at cell-cell-contacts, and thus all cells could be confirmed as endothelial cells. The localization of PECAM-1 corresponds to the one in primary HUVEC (compare Fig. 3.3).

Fig. 3.10: Immortalized HUVEC express endothelial marker PECAM-1. Immunofluorescence staining of immortalized HUVEC cells for the endothelial marker PECAM-1 (green) to verify endothelial origin. DNA was stained with Hoechst (blue). Oncoproteins used for immortalization are indicated. Immortalized cells were positive for PECAM-1, which localized at cell-cell-contacts between confluent cells. Absence of PECAM-1 signal in some cells can be explained by lack of confluence.

Results