Generating Bovine Induced Pluripotent Stem Cells (IPSCS) for Biotechnology and Preserving Genetics of Dairy and Beef Cattle

<p>NON-TECHNICAL SUMMARY:<br/> A recent breakthrough in stem cell biology is the generation of 'induced pluripotent stem cells (iPSCs)'. Induction of four genes Oct4 and Sox2 in conjunction with cMyc and Klf4 or Nanog and Lin28 could reprogram terminally differentiated cells into a pluripotent state indistinguishable from embryonic stem cells (ESCs). The conserved underlying mechanisms regulating this process has already led to the generation of iPSCs from different laboratory and farm animal species. In this proposal, our intention is to lay a preliminary foundation for the development of iPSC technology in domestic cattle. For animal agriculture: Generating a bovine iPSC-based platform would tremendously facilitate specific gene targeting of the bovine genome and the generation of genetically modified animals. Such dairy and beef cattle biotechnology is important to boost
U.S. agricultural production, improve global capacity to meet growing food demands, and foster innovation in fighting hunger and addressing food security. Especially at the present day as we face global climate change, introducing dairy and beef biotechnology can bring about sustainable productivity and economic vitality. Genetic engineering can improve food safety and control microbial contamination. Bovine iPS cell-based genetic preservation methods can also protect existing cattle genetic diversity.
APPROACH: 1. Generation of bovine iPS cells using lentiviral transduction of pluripotency factors: The objective of this experiment is to optimize methods for making bovine iPSCs from fibroblasts. Fibroblasts have been extensively used as a substrate to derive iPSCs from multiple species and will be grown either using embryonic tissues collected from the slaughter house, piece of the umbilicus after calving or from skin biopsy samples from adult cattle. Induction of fibroblasts to form iPSCs will be compared for two different systems: (a) Using a non-inducible set of 6 individual vectors for Oct3/4, Sox2, cMyc, Klf4, Lin28 and Nanog; (b) Using a tetracycline-inducible set of the same 6 genes and M2rtta. 2. Characterization of bovine iPS cells: The objective of this experiment is to validate the pluripotency of bovine iPSCs in vitro. After selection, bovine iPSC
colonies will be evaluated by using immunohistochemical labeling for pluripotency surface markers SSEA-1, SSEA-3, SSEA-4, TRA 1-60, and TRA 1-81 and transcription factors Oct3/4 and Nanog. In addition, an enzyme assay for alkaline phosphatase will also be performed. Chromosome numbers and integrity will be evaluated by staining metaphase spreads. In vitro differentiation of bovine iPS cells will be evaluated by generating embryoid bodies and testing both their spontaneous and directed differentiation potential into cell types of the three germinal layers. 3. Testing growth conditions required for the propagation of bovine iPS cells: The objective of this experiment is to determine the specific growth conditions required for self-renewal and pluripotency of bovine iPSCs. From preliminary studies, we have found that use of KSR medium with human FGF2 can maintain expanding bovine iPSC
colonies similar to human iPSCs. Previous experiments on bovine ES-like cells have documented the use of other factors in KSR medium including human LIF and bovine FGF2 for long-term culture. In this study, we will compare conditions using (a) human LIF, (b) bovine FGF2, and (c) both in combination, for the maintenance of bovine iPSCs. 4. Testing methods for optimal cryopreservation of bovine iPSCs: The objective of this experiment is to optimize for maximum freeze-thaw viability in storage of bovine iPSCs. Rho-kinase (ROCK) inhibitors that prevent dissociation-induced cell death during passage of human ESCs improved freeze-thaw viability in these cells. From literature on bovine ES-like cells, we believe that characteristics of bovine iPSCs will mirror human ESCs and iPSCs. However, there are no studies evaluating the cryopreservation properties of bovine ES-like cells. For bovine
iPSCs, we will examine the efficacy of different chilled freezing media (modified from human ESC protocols): (a) 90% KSR, 10% DMSO and (b) 90% KSR, 10% DMSO with Y-27632 on the freeze-thaw viability of different clones. In this experiment, freeze-thaw viability will be examined by comparing the number of iPSC colony forming units for each condition.
<p>PROGRESS: 2012/10 TO 2013/09<br/>Target Audience: US and NY state dairy and beef farmers would be the direct beneficiaries of this research effort. Results on bovine induced pluripotent stem cells (iPSCs) from this work will open a new chapter in dairy and beef cattle biotechnology providing a platform with potential for use in specific targeted genome manipulation and transgenesis for improving production traits and generating value-added dairy products. These genetic engineering strategies can improve profitability and reduce financial losses associated with both production and aspects of management. Finally, data from these studies will enable other researchers in the field of animal science to understand pluripotent stem cell biology and explore their field applications. Changes/Problems: Nothing Reported What opportunities for training and professional
development has the project provided? This project provided training for one graduate student. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We will proceed with experiments as proposed under our specific objectives for the next reporting period.
<p>PROGRESS: 2011/10/01 TO 2012/09/30<br/>OUTPUTS: Generation of pluripotent stem cells from somatic cells irrespective of sex, age and reproductive status of an animal offers numerous novel applications in domestic cattle. Reprogramming of somatic cells to what are called "induced pluripotent stem cells" (iPSCs) is mediated by the overexpression of factors - Oct4, Sox2, cMyc, Klf4, Lin28 and Nanog. During the first year of this work, we initiated experiments with an objective of optimizing methods for making iPSCs from bovine embryonic fibroblasts. Bovine embryonic fibroblasts were generated from a Day 30-35 old bovine embryo collected from an abattoir. Experiments are currently underway to test reprogramming of these cells using lentiviral vectors that code for the different factors. PARTICIPANTS: This project provided training for one graduate student. TARGET
AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
<p>PROGRESS: 2011/10/01 TO 2012/09/30<br/>OUTPUTS: Generation of pluripotent stem cells from somatic cells irrespective of sex, age and reproductive status of an animal offers numerous novel applications in domestic cattle. Reprogramming of somatic cells to what are called "induced pluripotent stem cells" (iPSCs) is mediated by the overexpression of factors - Oct4, Sox2, cMyc, Klf4, Lin28 and Nanog. During the first year of this work, we initiated experiments with an objective of optimizing methods for making iPSCs from bovine embryonic fibroblasts. Bovine embryonic fibroblasts were generated from a Day 30-35 old bovine embryo collected from an abattoir. Experiments are currently underway to test reprogramming of these cells using lentiviral vectors that code for the different factors. PARTICIPANTS: This project provided training for one graduate student. TARGET
AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.