Nuclear Transfer Embryonic Stem Cells: A Step Towards Therapy?
By Ann Carroll, PhD and Suzanne Kadereit, PhD

Background
Human embryonic stem cells pose great promise both for basic research and eventual use in the clinic (For background, see Embryonic Stem Cells).  

Embryonic stem cells are obtained from blastocysts, which are very early stage embryos containing about 100 cells. One potential problem for using these human embryonic stem cells for cell therapy is that the transplanted cells, which are genetically different from the patient, would be recognized as “foreign” and destroyed by the patient's immune system. (See Stem Cells and Immune Rejection.)

A possible way around this problem would be to obtain human embryonic stem cells by a technique known as nuclear transfer, formerly called therapeutic cloning. Instead of creating a blastocyst by fertilizing an egg with a sperm cell, as is done in the IVF (in vitro fertilization) procedure, nuclear transfer starts with an egg from which the nucleus (the cell's DNA) has been removed and replaced with the DNA from a body cell from the patient (see schematic on right). The use of the adult cell DNA ensures that all embryonic stem cells derived from the resulting blastocyst will be a match to the patient. For several years now, researchers have successfully used nuclear transfer techniques to establish embryonic stem cell lines in a range of animal species, but not, until recently, from the primate.

Establishment of Primate Nuclear Transfer Embryonic Stem Cell Lines
In December, 2007, scientists from the Oregon Primate Research Center, Oregon, USA, reported the derivation of two stem cell lines from blastocysts after nuclear transfer in the monkey (See Nuclear Transfer Stem Cells from Monkey). An earlier report in March 2004 by Woo Suk Hwang and colleagues that they had successfully established the first human embryonic stem cell line after nuclear transfer of adult human DNA into a human egg was unfortunately found to be fraudulent (See Science Magazine report).

The establishment of the first primate nuclear transfer embryonic stem cell line provided optimism that the technique will be feasible in humans and may ultimately be used for future clinical therapeutic cell replacement treatment free from immune rejection. However, significant hurdles remain. Most importantly, the procedure needs to be repeated in humans and the process optimized.

Remaining Questions and Hurdles for Generating Human Blastocysts by Nuclear Transfer:

• Need to refine and improve efficiency of nuclear transfer protocols
  
  Studies in different animal species have shown lower success rates for the derivation of embryonic stem cells from blastocysts created by nuclear transfer versus those blastocysts generated through normal fertilization. The reasons for this are not clear and need to be investigated.  
  
  To address this issue, researchers have begun to analyze the nuclear transfer procedures used in animal studies. In studies in non-human primates, for example, researchers noted that the method used for extracting the egg's DNA also resulted in removal of other egg cell components that are important for normal embryonic development. There also is evidence from some animal nuclear transfer studies that the newly inserted adult DNA does not behave properly. For nuclear transfer to work, unique components in the egg cell must allow for nuclear reprogramming of the newly inserted adult cell nucleus to occur. This ensures that it behaves as an embryonic cell nucleus would, and expresses, or activates, the required genes for smooth embryonic development.  
  
  
• Where will the human eggs needed for nuclear transfer come from?    
  For realization of future clinical application, it will be essential to address the question of how to procure the large numbers of human eggs that will be required.   With current efficiencies and methodology, several hundred eggs, and many human egg donors, would be needed for a single patient therapy. Also, human egg donation is not a simple or risk-free process for donors. Thus there is a real logistical and ethical dilemma about how to obtain adequate egg supplies for large-scale therapeutic applications. Therefore, some researchers are pursuing new avenues of research and are investigating potential alternative egg sources.
  
  
• Generation of egg cells from human embryonic stem cell cultures.
  Some researchers have described the generation of egg cells from laboratory cultures of mouse embryonic stem cells. The researchers are now studying whether these egg cells can be used in nuclear transfer experiments. If so, this may also be achieved in humans and could provide an important new source of human eggs.
  
  
• Egg cell progenitors in adult ovary.
  One research team found surprising evidence that adult mouse ovaries may contain a type of stem cell capable of forming new eggs throughout adulthood. If such cells could be found in humans, isolated and expanded in culture, this could open the door to a vast source of human egg cells.
  

Nuclear Transfer Embryonic Stem Cells in Animal Disease Models
In addition to the hurdles remaining for improvement of nuclear transfer procedures, there are other aspects of the technique to consider. In order to the use of embryonic stem cells for therapies, it is necessary to induce the cells to develop into stable cells that function normally and show the characteristics and properties of normal tissue cells. At this point, it is not clear if the mature cells derived from nuclear transfer embryonic stem cells are the same as mature cells derived from embryonic stem cells isolated from normal blastocysts (that is, those developed from fertilized eggs).

• Culture protocols by which the embryonic stem cells are pushed to become mature adult tissue type cells – such as functional blood cells, nerve cells, or insulin producing beta cells.  
  In one recent study, researchers reported that that they could push mouse nuclear transfer embryonic stem cells to develop into the type of nerve cell that is lacking in patients with Parkinson's disease. They injected those nerve cells into a mouse model of Parkinson's disease, and saw some improvements in the afflicted mice. This result provided evidence that the specialized cells generated from nuclear transfer embryonic stem cells in the laboratory are functional when transplanted into unhealthy animals. Generation of other mature cell types from nuclear transfer embryonic stem cells, for example insulin-secreting cells for cellular therapy for Type 1 diabetes, is proving more challenging.

• Combining embryonic stem cell therapeutic approaches with gene therapy.  
  Researchers have begun to explore possibilities of combining nuclear transfer with gene therapy. These combined techniques would repair a diseased gene by introducing a normal gene into the embryonic stem cells, and would produce therapeutic cells for a specific patient with a genetic disease.   A team at MIT combined nuclear transfer with gene therapy in a “proof of principle” experiment. They used nuclear transfer embryonic stem cells in which a specific gene was repaired to treat mice with a genetic deficiency. The team first derived the nuclear transfer embryonic stem cells from the mice with the genetic blood deficiency. They then used gene therapy to correct the mutation in those cells. Then they grew the “repaired” nuclear transfer embryonic stem cells and pushed them to develop into specific blood cells, which were transplanted into the afflicted mice. The mice were partially cured. This study emphasized the potential promise but also the complexities of such an approach and identified additional hurdles that need to be addressed before applying it to humans.
  

While much progress has been made in a short period of time (human embryonic stem cells were only isolated in 1998), numerous hurdles exist that must be addressed before heading into clinical applications for humans. Importantly, ongoing research has provided ‘proof of principle' that human therapy with embryonic stem cells matched to the patient is feasible.

Updated: February 14, 2008