Source: CORNELL UNIVERSITY submitted to
GENERATING BOVINE INDUCED PLURIPOTENT STEM CELLS (IPSCS) FOR BIOTECHNOLOGY AND PRESERVING GENETICS OF DAIRY AND BEEF CATTLE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0227259
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2011
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Project Director
Selvaraj, VI.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Animal Science
Non Technical Summary
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.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053999103050%
3057010102020%
3057410104030%
Goals / Objectives
Goal: To develop lentiviral-based reprogramming technologies for deriving bovine induced pluripotent stem cells (iPSCs). Specific Objectives: 1: To generate and characterize bovine iPSCs. 2: To optimize culture and cryopreservation protocols for bovine iPS cells. Expected Outputs: Authentic pluripotent embryonic stem cells (ESCs) for use in precisely targeted genome manipulation and controlled transgenesis are yet to be derived/generated for domestic cattle. As a result, advancement in both the safety and economic potential of dairy and beef cattle biotechnology for improving human food has reached an impasse. Moreover, this stumbling block has also curbed advancement of bovine reproductive technologies and genetic preservation beyond gametes. Therefore, generation of bovine induced pluripotent stem cells (iPSCs; with identical potential to ESCs) will provide a novel resource and open up unprecedented methods to address problems and facilitate improvements to husbandry, production, and genetic preservation of cattle. In this proposal, our intention is to lay a preliminary foundation for the development of iPSC technology in domestic cattle.
Project Methods
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.

Progress 10/01/11 to 09/30/14

Outputs
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 research training for one graduate student. How have the results been disseminated to communities of interest? Research methods and results were presented at the 68th Annual Convention of the National Association of Animal Breeders. This National Meeting was attended by 275 participants that included Animal Industry personnel, Scientists and Farmers. Information on bovine pluripotent stem cells was well received and beneficial outcomes that could result from this research were communicated to the attendees from across the United States. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Results from our studies addressed the specific objectives outlined under this grant. We have generated bovine induced pluripotent stem cells (iPSCs) and characterized them. Specific results show that numerous factors affect the method used to generate iPSC that include: starting cell type, gene introduction methods, culture conditions, growth factors required for maintain self renewal and method of passaging cells. Among all the different methods experimented, use of FGF2 in bovine iPSC medium was successful in maintaining cultures with reasonable self-renewal. Alternate methods using specific inhibitors only offered cells that could be passaged a few times. Cryopreservation of bovine iPSCs using conventional methods significantly affected their post-thaw viability. We optimized bovine iPSC cryopreservation using an fetal bovine serum based medium using a standard freezing container. Based on data obtained from this Hatch project, a continuation to advance these objectives has been recently funded by USDA-NIFA.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Pillai VV, Mo D, and Selvaraj V. (2014). Development of induced pluripotent stem cells (iPSCs) from cattle. 68th Annual Convention of the National Association of Animal Breeders. September 25-26, Green Bay, WI.


Progress 10/01/12 to 09/30/13

Outputs
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.

Impacts
What was accomplished under these goals? We have made significant progress towards accomplishing our specific objectives under this grant. Our studies showed that generation of bovine induced pluripotent stem cells (iPSCs) using lentiviral-mediated gene introduction is affected by numerous factors associated with the starting cell type. Rather than derivation, sustaining bovine iPSCs as pluripotent cells posed a challenge – as specific growth conditions for embryonic stem cells remain to be established for this species. In our studies, we identified that commonly used growth factors (leukemia inhibitory factor and basic fibroblast growth factor) and medium conditions do not support self-renewal and undifferentiated expansion of bovine iPSCs. In an alternate approach, rather than signaling for self-renewal, preventing signaling for differentiation is an approach taken to sustain rat embryonic stem cells. We are currently evaluating this method that uses three inhibitors (MEK inihibtor – PD184352, GSK3 inhibitor CHIR99021 and FGF receptor inhibitor SU5402) in the growth medium to prevent bovine iPSCs from differentiating. This approach has not been previously tested on bovine cells. Once growth conditions are established, we will progress to validation and set the stage for applications of these bovine iPSCs in livestock production.

Publications


    Progress 10/01/11 to 09/30/12

    Outputs
    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.

    Impacts
    The aim of this study is to generate bovine iPSCs and optimize conditions for their growth and propagation. In reprogramming trials, we have had success in generating bovine iPSCs that are positive for alkaline phosphatase. We are currently testing different growth conditions by adding individual growth factors in cell culture medium to identify the optimal factor and medium condition that would promote bovine iPSC expansion with minimal incidence of spontaneous differentiation. Growth factors that support pluripotency is bovine iPSCs have not been identified. Leukemia inhibitory factor (LIF) supports pluripotency in mouse iPSCs, fibroblast growth factor 2 (FGF2) supports pluripotency in human iPSCs. Therefore, we are testing the effect of these two growth factors in supporting undifferentiated expansion of bovine iPSCs. Once media conditions are standardized, we will progress to validating these cells through both molecular characterization (gene expression studies using quantitative PCR and immunohistochemistry) and developmental potential (directed differentiation and teratoma formation).

    Publications

    • No publications reported this period