For the first time, researchers at the University of Connecticut and the Institute of Zoology, Chinese Academy of Sciences have generated a stable line of embryonic stem cells from cloned cattle embryos that can make unlimited copies of themselves and can morph into cells for nearly all bovine body tissues and organs.
The research may offer a breakthrough tool for scientists studying the use of such cells to treat disease, because the study suggests that cattle stem cell lines may serve as a better model than those from other species for insight into human cell-based therapies.
“The bovine stem cells we generated are different from all previously reported lines in both morphology and marker expression patterns,” said Dr. Xiangzhong (Jerry) Yang, animal science professor and director of the University of Connecticut’s Center for Regenerative Biology (CRB). “This is the first report demonstrating morphology similar to those of established stem cells in humans and mice.”
The researchers, led by Dr. Yang, Dr. Cindy Tian, also with the CRB, and Dr. Enkui Duan of the Institute of Zoology of the Chinese Academy of Sciences in Beijing, China, report their findings in the March 2005 issue of the journal Biology of Reproduction.
Their results come at a time when international research on embryonic stem cells is picking up speed. Because embryonic stem cells can self renew and grow into virtually any cell in the body, they offer a potentially unlimited supply of specific cells for replacement therapy in degenerative disorders such as diabetes and Parkinson’s disease.
But for embryonic stem cell-based therapy to become a clinical reality, translational research involving nonhuman species is essential. Stable embryonic stem cell lines have been established in a few species such as mice and monkeys, but generating a stable bovine embryonic stem cell line has proven difficult.
Results have varied in five reported attempts to generate bovine embryonic stem cell lines that are pluripotent ? capable of proliferating indefinitely and differentiating into a wide variety of cell types. Also, none of these earlier studies of bovine embryonic stem cells found any cell surface markers of proteins required for growth that are normally associated with embryonic stem cell lines generated from other species.
Yang, Duan and their colleagues began their study by creating three separate bovine embryonic stem cell lines - one obtained from an in vitro fertilized embryo and two others from nuclear transfer embryos.
The in vitro technique is simple enough; a test-tube embryo is formed after an egg is fertilized in the laboratory. Nuclear transfer involves removing the nucleus from an unfertilized egg and replacing it with the DNA taken from a cell elsewhere. Using chemicals and electric shock, the egg containing the transferred DNA is then induced to divide until it forms a hollow, fluid-filled cavity surrounded by a layer of cells. This embryo, known as a blastocyst, holds the precursors to embryonic stem cells.
The researchers cultivated the three stem cell lines in the laboratory growing them into large, multicellular colonies resembling mouse and human embryonic stem cells. Throughout this period they also studied the makeup of the cells by washing the clusters with chemicals that prompted stains, or cell surface markers, associated with pluripotency in embryonic stem cells.
"The embryonic stem cell marker staining patterns of the two nuclear transfer cell lines are identical to those of the in vitro fertilized blastocysts, yet different from all previously reported bovine embryonic stem cells which were negative for these staining data," Yang said.
"The fact that the embryonic stem cell-specific marker staining pattern is very similar to the human embryonic stem cells suggests that bovine embryonic stem cells may serve as a better model than mouse embryonic stem cells for human embryonic stem cell regeneration studies," Yang said.
After sustaining continuous undifferentiated growth in the colonies for over a year, the researchers found that the cells maintained an ability to form globular clusters called embryoid bodies representative of the body’s three broad cell lines (the endoderm, mesoderm and ectoderm) both in vivo and in vitro, underscoring their ability to morph into cells for nearly all body tissues.
"The differentiation of our cells in vitro (in glass, outside the animal) into many specific cell types, representative of all three embryonic germ cell layers, suggests that these cells are indeed pluripotent," said Yang. “Additional studies to demonstrate the ability of these cells to form germ lines in vivo will further support our characterization of pluripotency.”
If researchers can find a way to direct the growth of embryonic stem cells in the lab, they might be able to engineer healthy cells to treat a wide range of ailments. This will require efficient methods to grow tissue specific cells as embryonic stem cells differentiate.
Using enzymes to break up colonies and isolate individual cells for new colony formation instigates this process in mouse, monkey and human cell lines. In this study, researchers found that enzymes failed to break apart the bovine embryonic stem cell colonies and induce spontaneous differentiation.
"Previous studies have shown that the embryonic stem cells’ self-renewal pathway differs between species," Yang said. "Further studies are needed to understand why bovine pluripotent embryonic stem cells are not amenable to enzymes."
About UConn’s Center of Regenerative Biology (CRB):
The CRB is UConn’s world-class powerhouse in the research field of stem cells cloning, regenerative biology and medicine. Over the past eight years, UConn has generated a world reputation in the area of animal cloning and related reproductive biotechnologies. In 1998, Dr. Yang’s lab produced the first cloned male animal (bull) in the world. In June 1999, Amy, the calf was born, the first cloned animal from an adult farm animal in North America. Dr. Yang’s work on telomere and telomerase reprogramming in cloned animals was selected as one of the top 10 breakthroughs of the year 2000 by the journal Science. In 2004, Yang’s laboratory reported the production of the first second-generation clones in the world in a non-rodent species. UCONN’s research breakthrough combined nuclear transfer with genetic engineering to produce animal clones for human disease studies or transgenic animals to produce pharmaceutical proteins.
Recognizing these achievements and the research potential of animal cloning and its many ramifications such as stem cells and cell/tissue regeneration has for business opportunities in agriculture and bio-medicine, UConn constructed the $10 million Advanced Technology Laboratory building which hosts the CRB laboratories and other core facilities. Six extremely well qualified faculty members have been recruited to the Center, which opened and dedicated in 2003. These faculty's research areas are well-focused in the field of regenerative biology and add to the existing strong UCONN embryo biotechnology program with the following complementary expertise: nuclear transfer cloning and knockout technology, molecular embryology, embryonic and adult stem cells development and characterization, mechanisms of stem cell maintenance and differentiation, gene expression and cell/tissue engineering.
The ultimate objective of the CRB is to investigate areas of basic science that might lead to the therapeutic production of new cell types, tissues or organs as potential replacements for diseased tissues commonly found in disorders such as diabetes, Parkinson’s disease, multiple sclerosis, muscular dystrophy and many cancers.