Elsevier

Theriogenology

Volume 77, Issue 1, 1 January 2012, Pages 220-228.e2
Theriogenology

Research article
Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid

https://doi.org/10.1016/j.theriogenology.2011.09.022Get rights and content

Abstract

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.

Introduction

Gene banking of cells and tissues using cryopreservation is an important and useful approach for genetic preservation of valuable domestic cat breeds and for conservation management of endangered wild feline species [1]. Although, cryopreservation of gametes is the most useful method of supporting endangered species breeding programs [2], [3], collection of gametes from these species for assisted reproductive technology (ART) is often difficult. Recent advances in embryonic stem (ES) cell technology have provided an alternative approach, since ES cells can differentiate to gametes in vivo and therefore have the potential to provide a source of gametes for in vitro for embryo production. In addition, they can also be used as a donor cell for nuclear transfer (NT) and can be readily cryopreserved for gene banking [4].

The snow leopard (Panthera uncia) is a large cat that lives in the mountain ranges of Central Asia, between 3,000 and 5,500 m (9800 and 18,000 ft) above sea level [5]. Although the secretive nature of the snow leopard makes an accurate population census difficult, estimates suggest that only between 3,500 and 7,000 snow leopards still exist, making them an endangered species with numbers on the decline [6].

Endangered felid species are often difficult to breed both in captivity and under natural conditions. One of the most important reasons for infertility or subfertility is decreased genetic diversity caused by inbreeding, due to genetic bottle-necks because of geographic isolation and population contraction [7]. Consequently, there has been increasing interest in techniques for maintaining genetic diversity of endangered wild felids [8].

Pluripotent stem cells differentiate into all the cell types in the body, while retaining the capacity for indefinite self-renewal [9]. These cells have great potential for application in regenerative medicine, assisted reproductive technologies, development of new biotechnologies, and drug development [10]. Pluripotent stem cells have traditionally been derived from embryos, which are destroyed in the process, raising ethical and moral concerns for the derivation of stem cell lines in humans and also in endangered species. For species in which embryos are particularly difficult to obtain, such as endangered species, this approach also faces logistical concerns, as the supply of embryos in wild felids for isolation of ES cells is limited. Induced pluripotent stem (iPS) cells, which are derived from somatic tissue are a potentially useful alternative to ES cells.

Production of iPS cells was first reported by Takahashi and Yamanaka [11] using viral transduction of mouse fibroblasts to screen a combination of 24 candidate genes with putative roles in pluripotency. They found that four transcription factors (OCT3/4, SOX2, KLF4 and cMYC) were required to reprogram mouse embryonic fibroblasts (mEFs) and adult tail tip fibroblasts to iPS cells, that were almost indistinguishable in morphology from mouse embryonic stem (mES) cells [12], [13]. Subsequently, iPS cells have been derived from the somatic cells of rodents, primates, dogs, sheep, horses, pigs and cattle [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], but there are no reports of iPS cells from any felid or endangered species.

To investigate whether this technology could be applied to endangered species, we attempted generation of iPS cells from snow leopard ear fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding either four (OCT4, SOX2, KLF4 and cMYC) or five (OCT4, SOX2, KLF4, cMYC and NANOG) human transcription factors. Our hypothesis was that inclusion of NANOG to the cocktail, which is critical for pluripotency in large animals [24], would be required to generate snow leopard iPS cells. Our aim was to derive and characterize iPS cells from snow leopard fibroblasts using retroviral vectors and to examine their differentiation potential both in vitro and in vivo.

Section snippets

Animals

Animal handling and experiments conformed to the code of practice of the Australian National Health and Medical Research Council (2004) and were approved by Monash University Animal Experimentation Ethics Committee.

Isolation of snow leopard ear fibroblasts

Tissue samples were collected from the ear pinnae of snow leopard, which had died of natural causes or were euthanized due to health-related problems identified by a zoo veterinarian. All samples were donated by Mogo Zoo (Australia).

Adult dermal fibroblasts cell lines were derived

Generation of snow leopard iPS cells

Transduction efficiency of the retroviral transfection using pMX-GFP transgene expression, averaging 96% from three repeated experiments, are shown in Table 1. The reprogramming efficiency for initial colony formation following five factor induction was 0.000517%, compared with 0.000308% for four factor induction. Only five factor induction resulted in colony survival (80%) beyond P4. Three day post-infection, the appearance of compact colonies was noted (Fig. 1A). The colonies that were

Discussion

Since the initial generations of murine iPS cells [11], there have been numerous attempts to derive iPS cells from a range of other species. However with the exception of rodents, primates and rabbit complete reprogramming of somatic cells has not been reported. To date the lack of silencing of inserted transgenes has been a hallmark of iPS cells in large animals, including dog, sheep, monkey, horse, pig and cattle [18], [20], [21], [22], [23], [29], [30], [31], [32].

Preliminary experiments

Acknowledgments

We thank Sally Padey, (Director) and Hannalie Vander Merwe, (Officer in charge) of Mogo Zoo in New South Wales (Australia) for their generous support of this project by providing tissue samples. We also thank to all the staff at Mogo Zoo, veterinarians Dr. Mary Atkinson and Dr. Peter Atkinson and veterinary pathologist Dr. Mark Williamson (Gribbles Pathology, Australia). Rajneesh Verma acknowledges a Ph.D. scholarship awarded by Professor Bryan Williams and the Monash Institute of Medical

References (32)

  • D.R. Bainbridge et al.

    Potential of assisted breeding techniques for the conservation of endangered mammalian species in captivity: A review

    Vet Rec

    (1998)
  • D.E. Wildt et al.

    Assisted reproduction for managing and conserving threatened felids

    Int Zoo Yearbook

    (1997)
  • L. Wei et al.

    The complete mitochondrial genome structure of snow leopard Panthera uncia

    Mol Biol Rep

    (2009)
  • Sassa Y., Yamamoto H., Mochizuki M., Umemura T., Horiuchi M., Ishiguro N., et al. Successive deaths of a captive snow...
  • Y. Toyooka et al.

    Embryonic stem cells can form germ cells in vitro

    Proc Natl Acad Sci U S A

    (2003)
  • G.B. Zhou et al.

    In vitro derivation of germ cells from embryonic stem cells in mammals

    Mol Reprod Dev

    (2010)
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