Programming of bovine stem cells from adult cells may help improve human and animal health

Yong Tang (pointing at screen) and his graduate students examine human iPSCs they generated using "super" OCT4 fusion proteins.
Yong Tang (pointing at screen) and his graduate students examine human iPSCs they generated using “super” OCT4 fusion proteins.

Young Tang, assistant professor in the Department of Animal Science, is intrigued by unanswered questions. Tang is currently engaged in an intricate study, a large-scale next-generation genomic analysis that may help improve both human and bovine health.

The study involves induced pluripotent stem cells (iPSCs), which hold great promise for regenerative medicine. Human pluripotent stem cells are self-replicating and can be developed into all other cells types in the body, which could provide a source of replacement cells for those lost to damage or disease. The most well-known type of pluripotent stem cell is the embryonic stem cell, but due to the controversy surrounding this type of stem cell, scientists have been seeking alternatives. iPSCs are a type of pluripotent stem cell that can be generated directly from adult cells such as skin cells. This technology was developed at Shinya Yamanaka’s laboratory in Japan.

Four genes—OCT4, SOX2, KLF4 and cMYC—are commonly introduced into mature cells to produce iPSCs. “These genes encode proteins, called transcription factors, that regulate cellular gene expression,” says Tang. “During this reprogramming process, these four factors work together to change the cellular identity from differentiated somatic, or specialized, cells to pluripotent stem cells.”

Although this technology has been developed for human, mouse and a few other species, efforts have been unsuccessful for bovine species despite numerous attempts.

Large livestock iPSC’s are more closely related to humans’ than are mouse iPSCs. Therefore, bovine iPSCs are more useful for regenerative medicine studies, particularly for pre-clinical animal models of human diseases. In addition, bovine iPSCs could be used in agricultural research to screen for superior traits that may lead to improved reproduction and disease resistance in cattle. Technology for the development of bovine iPSCs would provide invaluable cell resources for these types of research.

With regard to the lack of success so far in developing bovine iPSCs, Tang says there may be a bovine-specific difference controlling the reprogramming process. He is examining the sequence of events to determine if there is a gene or signaling pathway in bovine cells that could be activated or suppressed during this process.

“This would provide highly valuable data to improve bovine iPSC generation and applications,” he says.

Bovine-specific pluripotent gene- controlled GFP reporter is activated in a human pluripotent stem cell colony.
Bovine-specific pluripotent gene-controlled GFP reporter is activated in a human pluripotent stem cell colony.

The established iPSC reprogramming process is not efficient, taking weeks to complete. It can take three to six weeks to successfully program one human iPSC colony from 1,000 to 100,000 starter cells. Tang is working to develop tools that facilitate the development and identification of the true iPSC cells during the nuclear reprogramming of somatic cells. One effort is to create a “super protein,” a fusion of  two proteins used in human iPSC cell generation. By using part of the MYC protein, called the transactivation domain (TAD), that strongly activates the expression of MYC targeted genes, his group generated MYC-TAD-OCT4 fusion proteins to reprogram primary human cells. Tang says, “This process showed dramatic enhancement of iPSC colony generation efficiency and shortened reprogramming dynamics.”

Tang discovered that using green fluorescent protein (GFP) reporters, which are widely used in studies of human pluripotent stem cell establishment, may be useful in identifying successfully generated bovine iPSC cells. The DNA of genes used in the iPSC induction process are injected with GFP, which fluoresces to “report” the activation of these genes in successfully reprogrammed cells. His research team developed bovine gene-specific GFP reporters with demonstrated activity in both human and mouse pluripotent cells, which can be used to identify and isolate the successfully reprogrammed pluripotent stem cells in bovine iPSC induction.

In a separate collaborative study. Tang and Antonio Garmendia, professor in the Department of Pathobiology and Veterinary Science, are seeking ways to combat porcine reproductive and respiratory syndrome virus (PRRSV), a devastating global swine disease.

PRRSV attacks lung macrophages, a type of immune cell that helps eradicate foreign pathogens. PRRSV can dramatically reduce the general health of the animal by causing symptoms such as high fever, coughing and diarrhea; increase the likelihood of secondary infections; and adversely affect reproduction, resulting in miscarriages and high mortality rates of newborn piglets.

No single vaccine has been effective for PRRSV, as the virus contains many subtypes. Tang and Garmendia are seeking a new approach to combat the virus.

“We are collaborating in a research effort aimed at identifying cellular events essential for PRRSV infection during the virus-host interaction that can be blocked by the use of small chemicals, thus curtailing infection,” Garmendia explains. “Reducing PRRSV susceptibility would have a positive impact for the swine industry.”

These studies are supported by multiple grants from NIFA/USDA; Atomwise Inc.; Program in Innovative Therapeutics for Connecticut’s Health (PITCH); UConn CAHNR Capacity Competitive Funds; and UConn’s Research Excellence program.

By Kim Colavito Markesich