Derivation and characterization of cell cultures from the skin of the Indo-Pacific humpback dolphin Sousa chinensis

Abstract

The marine mammalian Indo-Pacific humpback dolphin, once widely lived in waters of the Indian to western Pacific oceans, has become an endangered species. The individual number of this dolphin has significantly declined in recent decades, which raises the concern of extinction. Direct concentration on laboratorial conservation of the genetic and cell resources should be paid to this marine species. Here, we report the successful derivation of cell lines form the skin of Indo-Pacific humpback dolphin. The cell cultures displayed the characteristics of fibroblast in morphology and grew rapidly at early passages, but showed obvious growth arrest at higher passages. The karyotype of the cells consisted of 42 autosomes and sex chromosomes X and Y. The immortalized cell lines obtained by forced expression of the SV40 large T-antigen were capable of proliferation at high rate in long-term culture. Immortalization and long-term culture did not cause cytogenetically observable abnormality in the karyotype. The cell type of the primary cultures and immortalized cell lines were further characterized as fibroblasts by the specific expression of vimentin. Gene transfer experiments showed that exogenetic genes could be efficiently delivered into the cells by both plasmid transfection and lentivirus infection. The cells derived from the skin of the Indo-Pacific humpback dolphin may serve as a useful in vitro system for studies on the effects of environmental pollutants and pathogens in habitats on the dolphin animals. More importantly, because of their high proliferation rate and susceptibility to lentivirus, these cells are potential ideal materials for generation of induced pluripotent stem cells.

Establishment and characterization of pygmy killer whale (Feresa attenuata) dermal fibroblast cell line

“…Primary cells lifespan is limited to several passages and limits the long-term availability of these cells for further research. Similarly, Jin et al, [ 16 ] found that fibroblast cell cultures morphology and growth noted rapid at early passages, but later showed obvious growth arrest at higher passages. To overwhelm this issue, gene transfer of SV40 early regions has been widely used to escape the Hayflick limit [ 17 , 22 ].…”

“…It was previously reported that the normal diploid somatic cells of cetacean species contain 21 or 22 pairs of chromosomes [ 8 ], which can show multiple stages of chromosomes. Similarly, Jin et al [ 16 ] showed 44 chromosomes in Chinese white dolphin fibroblast cells. Cetacean chromosomes numbers often have 42 to 44 and may appear metacentric, submetacentric, telocentric and sub-telocentric [ 11 ].…”

“…In the present study, we developed dermal fibroblast cell line (PKW-LWH) and established fibroblast cell line (PKW-LWHT1) by transfecting SV40 T antigen gene into a primary cell line. A similar approach was applied to develop primary cell culture line from humpback whale skin tissue [ 15 ] and to establish the fibroblast cell line from Chinese white dolphin [ 16 ], and skin fibroblast from another cetacean species, Yangtze finless porpoise [ 11 ].…”

“…Whereas, findings of Burkard et al [ 15 ] previously described that established epidermal cell cultures exhibit squamous cells morphology, express vimentin, and grow very well until passage thirty. The growth rate of the primary cells culture with higher passages slows down, presumably as a result of senescence [ 16 ]. To overcome cell culture morphological modulation and growth arrest, SV40 transfection induces forced expression of large T-antigen, leading to the production of cell lines and enabling them capable of continuous proliferation [ 17 ].…”

Establishment of the first humpback whale fibroblast cell lines and their application in chemical risk assessment

“…G r o w t h o f H u W a 1 a n d 2 Jin et al (2013) reports the establishment of humpback dolphin fibroblast cultures and assessed 3-5 days of proliferation until cells reached confluency when passaged 1:3. The same study observed reduced proliferation starting from P10 and finally senescence with P17.…”