- Efficient and reproducible generation of high-expressing, stable human cell lines without need for antibiotic selection
Primary amniocytes can be efficiently transformed by adenoviral E1-functions
In order to test if primary amniocytes can be used to establish permanent, therapeutic protein expressing cell lines, amniocytes were cotransfected with the E1-expressing plasmid pGS119 containing E1A-, E1B- and pIX-functions plus the plasmid pGS116 expressing the human alpha-1 antitrypsin (hAAT) glycoprotein.
The primary amniocytes used here originated from a single amniocentesis sample. About 20–30 days after transfection of approximately 1 × 106 cells, numerous transformed cell colonies appeared on all dishes, and cells on the dishes were expanded as 10 pools (Z171-1A, B – Z171-5A, B). Non-transformed cells stop growth already at early passages and in approximately passage 10–15 mainly transformed cells can be detected on the dishes. Since transfection efficiency of primary cells is less than 1% (data not shown) the efficiency of transformation is very high, however, because cells were passaged twice the number of independently transformed clones could not be determined. Such high frequencies were obtained without difference in numerous transfections using different cells from several amniocentesis (data not shown).
Expression of the E1 proteins was analyzed by Western Blot using protein extracts of different cell pools. Like HEK293 cells used as positive control, all cell pools express both the E1A- and 21kD-E1B proteins, although in different amounts and ratios (Fig. 1).
Primary amniocytes show variable morphologies. In contrast, transformed cells do appear smaller and show more uniform morphologies. In very early passages the morphologies in different cell pools are quite similar but change during further cultivation. Most transformed cells go through mild crisis and decelerate growth but recover during only few passages.
Transformed amniocytes express and secrete hAAT
As described above, the primary amniocytes were cotransfected with a hAAT-expressing plasmid. hAAT is a major human serum protein which is predominantly produced and efficiently secreted from hepatocytes. Thus we tested for secreted (Fig. 2a) and intracellular (Fig. 2b) hAAT in different cell pools. Using Western Blot analyses we were able to detect hAAT expression in 6 out of 10 different cell pools. The amount of protein loaded per lane for the intracellular and secreted protein corresponds to 8 × 104 cells and 4 × 103 cells, respectively. These results suggest that hAAT is very efficiently secreted from the cells since only small amounts of the protein were intracellularly located.
Amniocyte cell lines show high and long-lasting hAAT expression
The above results show that both plasmids integrated upon cotransfection and cells express proteins from both plasmids. The lack of selective pressure during cell passaging most likely does not influence expression of E1-functions since their continuous presence is expected to be crucial to maintain the transformed phenotype of the cells. Therefore, we tested for the stability of expression of hAAT during multiple passages.
Six Z171 cell pools showed expression of hAAT in early passages and were thus further cultivated up to 35–50 passages. In different passages 7 × 105 cells were plated, the supernatants were collected and the amounts of secreted hAAT were quantitated. In figure 3 the amount of hAAT in 6 different cell pools is depicted and shows that 4 out of 6 cell pools show high and long-lasting hAAT expression up to 6 μg/ml. Only in two cell pools the hAAT expression drops drastically to almost not detectable expression levels after 30 passages. Two cell pools (Z171-5A and Z171-5B) stably express up to 8 pg hAAT per cell and day for more than 50 passages without any antibiotic selection.
Since each cell pool is derived from numerous different transformation and integration events and thus contains many genetically different cell lines, we performed single cell cloning on cell pools Z171-5A and Z171-5B by limited dilution in 96-well plates. Thirteen single cell clones originating from Z171-5A and 25 single cell clones originating from Z171-5B were expanded and tested for hAAT expression (data not shown). Figure 4 shows long term expression of hAAT of 3 clonal cell lines each originating from pool Z171-5A and Z171-5B, respectively. Over 60 passages all 6 clonal cell lines express hAAT; in 2 clonal lines maximum hAAT levels reach 27 and 30 pg/cell/day.
hAAT expressed in amniocyte cell lines is glycosylated and sialylated
Human alpha-1 antitrypsin is a 396- amino acid serum glyoprotein and contains three carbohydrate side chains N-linked to asparagine residues. Analyses of the carbohydrate composition revealed two main A- and B-types oligosaccharide chains (see Fig 5c) in 2:1 ratio [22,23]. Glycosylation of hAAT does not seem to be important for the formation of a biologically active conformation with elastase and thus for activity of the protein . However, glycosylation seems to play a crucial role in stability of hAAT in the serum [25,26] and in secretion from hepatocytes .
In order to test if amniocyte cell lines produce glycosylated hAAT we treated the cell culture supernatants of Z171-5A (Fig. 5) and Z171-5B (data not shown) with PNGaseF, followed by hAAT-specific Western Blotting. Since PNGaseF cleaves between the innermost GlcNAc and asparagine residue of oligosaccharides from N-linked glycoproteins (see Fig. 5c) we expected a shift in molecular weight if PNGaseF can hydrolyze N-glycan chains from hAAT. For control we used hAAT purified from human serum. Digestion with increasing amounts of PNGaseF results in a clear shift in molecular weight (Fig 5a). Moreover, appearance of three distinct bands clearly suggests the presence of three glycan chains in the recombinant product. There is no difference in size and number of different protein bands when compared to hAAT purified from human serum.
All galactoside residues in plasma derived hAAT are known to be linked with sialic acid. To test for this feature in hAAT expressed in amniocyte cell lines, protein secreted into the cell culture medium of Z171-5A was digested with Neuraminidase followed by hAAT-specific Western Blotting (Fig. 5b). Comparable to plasma derived hAAT, the hAAT produced in Z171-5A showed a shift in size when treated with Neuraminidase.
These results indicate that hAAT produced in human amniocyte cell lines are fully glycosylated and sialylated.
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