Using assisted reproductive technologies for the conservation of endangered wild cats
thesisposted on 28.02.2017 by Verma, Rajneesh
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The advent of induced pluripotency has opened alternate avenues to produce ES-like cells from somatic cells, that provide a unique tool to elucidate both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production of and research on embryonic stem cells difficult, we first investigated the appropriate species transcription factors to use for induction of pluripotency in wild cats. Octamer binding transcription factor 4 (Oct4), also known as POU5F1, is a protein critically involved in stem cell renewal and pluripotency and a key factor involved in reprograming. To identify the suitability of using either mouse or human Oct4 constructs, which pre-existed in the laboratory to derive induced Pluripotent Stem Cells (iPSC) from endangered cats, the coding sequence of domestic cat Oct4 was compared with that from other known species. Cat Oct4 protein has 94.44% and 83.33% sequences similarity compared with human and mouse Oct4, respectively. (Publication 1- paper submitted). In addition, the coding sequence for Oct4 were successfully amplified and cloned from fibroblasts of the three wild cats and compared in silico. Bioinformatics analysis revealed that although the sequences of coding regions vary slightly between snow leopard, Bengal tiger and jaguar, the predicted theoretical protein consist of 360 amino acids and is identical between these species. By contrast to the exons, introns and 5’ region close to the exon1 are less conserved in the three species. A phylogenetic tree was constructed using the neighbour-joining method based on the alignment of the coding sequences of Oct4 gene. As Oct4 is usually expressed in oocytes/early embryos, which are difficult to obtain from endangered species, the verification of the mRNA sequence of the gene posed obvious challenges. In conclusion, this study is the first to describe the molecular cloning and bioinformatics analysis of snow leopard, Bengal tiger and jaguar Oct4 gene (Publication 1- submitted). Information on the specific sequences of Oct4 in these endangered big cats will be useful to understand and manipulate pluripotency in these species. This has an exciting potential to contribute to real-life species preservation, particularly for endangered felids. Therefore we determined to use human sequences for induction of pluripotency in snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four human 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). We hypothesized the addition of NANOG, another transcription factor critically linked to Oct4 mediated pluripotency, to the transfection cocktail would enhance generation and maintenance of stable iPSC colonies, which formed as early as Day3 (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. Reverse Transcriptase PCR (RT-PCR) also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPSC from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; signifying reprogramming of the endogenous pluripotent genes had occurred. When injected into severe combined immuno-deficient (SCID) mice, snow leopard iPSC formed teratomas containing tissues representative of the three germ layers. In conclusion, this was the first report on derivation of iPSC from an endangered felid and the first report on induced pluripotency in felid species and it demonstrated the addition of NANOG to the reprogramming cocktail was essential for derivation of stable iPSC lines in this felid (Publication 2- published). Next, to examine whether the importance of Nanog for generation of iPSC was restricted to snow leopard or more generally applicable to wild felids globally, we studied 3 geographically diverse and evolutionarily divergent felids from Asia (Bengal tiger, Panthera tigris), Africa (serval, Leptailurus serval), and the Americas (jaguar, Panthera onca). Dermal fibroblasts were transduced with genes encoding the human transcription factors OCT4, SOX2, KLF4, and cMYC with or without NANOG. Both four- and five-factor induction resulted in colony formation at day 3 in all three species tested; however, we were not able to maintain colonies that were generated without NANOG beyond passage (P) 7. Five-factor induced pluripotent stem cell (iPSC) colonies from wild cats were expanded in vitro on feeder layers and were positive for alkaline phosphatase and protein expression of OCT-4, NANOG, and stage-specific embryonic antigen-4 at P4 and P14. Reverse-transcription polymerase chain reaction confirmed that all five human transgenes were transcribed at P4; however, OCT4, SOX2, and NANOG transgenes were silenced by P14. Endogenous OCT4 and NANOG transcripts were detected at P4 and P14 in all cell lines confirming successful reprogramming. At P14, the iPSC from all three species remained euploid and differentiated in vivo and in vitro into derivatives of the three germ layers. Our study demonstrated unequivocally that iPSC from the three felids examined all required the addition of Nanog to the reprogramming cocktail to ensure derivation of stable iPSC lines. Interestingly iPSC from all three species silenced the reprogramming transgenes (OCT4, SOX2 and NANOG), but not the transgenes implicated in proliferation (KLF4 and cMYC) (Publication 3- published). This thesis investigated induced pluripotency in endangered felids and reports successes in effectively inducing pluripotency in four endangered wild cats from across the globe and importantly identifies Nanog as an essential factor in the reprogramming cocktail. Efficient production of iPSC from endangered felids creates a unique opportunity to preserve these species using these iPSC in future gamete production, nuclear transfer and embryo complementation.