Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer

Abstract

Background: Somatic cell nuclear transfer (SCNT) provides an appealing alternative for the preservation of genetic material in non-domestic and endangered species. An important prerequisite for successful SCNT is the availability of good quality donor cells, as normal embryo development is dependent upon proper reprogramming of the donor genome so that embryonic genes can be appropriately expressed. The characteristics of donor cell lines and their ability to produce embryos by SCNT were evaluated by testing the effects of tissue sample collection (DART biopsy, PUNCH biopsy, post-mortem EAR sample) and culture initiation (explant, collagenase digestion) techniques.

Results: Differences in initial sample size based on sample collection technique had an effect on the amount of time necessary for achieving primary confluence and the number of population doublings (PDL) produced. Thus, DART and PUNCH biopsies resulted in cultures with decreased lifespans (<30 PDL) accompanied by senescence-like morphology and decreased normal chromosome content (<40% normal cells at 20 PDL) compared to the long-lived (>50 PDL) and chromosomally stable (>70% normal cells at 20 PDL) cultures produced by post-mortem EAR samples. Chromosome stability was influenced by sample collection technique and was dependent upon the culture's initial telomere length and its rate of shortening over cell passages. Following SCNT, short-lived cultures resulted in significantly lower blastocyst development (< or = 0.9%) compared to highly proliferative cultures (11.8%). Chromosome stability and sample collection technique were significant factors in determining blastocyst development outcome.

Conclusion: These data demonstrate the influence of culture establishment techniques on cell culture characteristics, including the viability, longevity and normality of cells. The identification of a quantifiable marker associated with SCNT embryo developmental potential, chromosome stability, provides a means by which cell culture conditions can be monitored and improved.

Figure 1

Length of time to reach primary confluence for cattle and gaur cell cultures. The numbers of days necessary for the dissociated cells to become confluent monolayers are shown for the sample collection (DART, PUNCH, EAR/SKIN) and cell dissociation (EXPL, COLL) treatment groups. Time to reach confluency varied according to the sample collection and cell dissociation techniques used.

Figure 2

Replicative lifespan of cattle and gaur cell cultures. The numbers of population doublings to reach senescence are shown for the sample collection (DART, PUNCH, EAR/SKIN) and cell dissociation (EXPL, COLL) treatment groups. Differences in the number of population doublings prior to senescence were observed between the sample collection techniques and only between cell dissociation techniques when short-lived cultures were produced.

Figure 3

Morphology of gaur cell cultures during serial cultivation in vitro. Phase contrast micrographs of gaur EAR fibroblast cultures from early and late passages demonstrate the changes in cell morphology that occurred with increased passages. Note the morphology (enlarged and flattened cells) characteristic of senescent cultures in (I) and (J). No major effects of cell dissociation techniques were observed in these long-lived cell cultures.

Figure 4

Morphology of gaur cell cultures during serial cultivation in vitro. Phase contrast micrographs of gaur DART and PUNCH fibroblast cultures from early and later passages demonstrate changes in cell morphology. Senescent morphology (enlarged and flattened cells) was observed within the first few passages as shown in (I) to (L). Effects of cell dissociation techniques were apparent in these short-lived cell cultures.

Figure 5

Chromosome content of cell cultures during serial cultivation in vitro. Percentages of chromosomally normal cells (% Normal Cells) were assessed for (A) cattle and (B) gaur cell cultures. A decrease in chromosomally normal cells occurred in all cultures with increased passages. Differences in the initial percentage and the rate of decline of chromosomally normal cells were observed in the different treatment groups. Effects of sample collection techniques were clearly evident in the gaur cell cultures.

Figure 6

Relative telomere lengths of cell cultures during serial cultivation in vitro. Relative telomere lengths (RTL) were assessed for (A) cattle and (B) gaur cell cultures. A decrease in RTL occurred in all cultures with increased passages. Effects of sample collection and cell dissociation techniques were not clearly evident.

Figure 7

Morphology of cattle EAR cell preparations for SCNT. Phase contrast micrographs of (A) early passage (P-5) and (B) late passage (P-30) fibroblast cultures prepared for SCNT demonstrate changes that occur following prolonged growth in vitro. A marked increase in cells with enlarged size and abnormal morphology was observed in the late passage culture.