Establishment of human embryonic stem cell lines using re-constructed human embryos derived from polyspermic eggs

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Public Abstract Using human embryos produced from in vitro fertilization laboratories is always a major ethical concern. There is a great resource neglected in this field, which is the eggs containing two sperms. This type of egg, called tripronuclear zygotes, will be routinely discarded in the in vitro fertilization laboratory. Approximately 7% of fertilized eggs contain more than one sperm, with tripronuclear zygotes as a most common phenomenon. There is some evidence that tripronuclear zygotes could develop into normal embryos. Recently, a normal, live human birth was achieved by removing the extra male pronucleus in the zygote. Previously, in pigs, the normal piglets were delivered by transferring the polyspermic eggs confirmed by microscope. The principal investigator for this proposal also reported in 2001 that polyspermy in pigs could be a physiological phenomenon if extra sperm did not affect the embryonic genome. Therefore, in this proposal, the extra male pronucleus from the tripronuclear zygotes will be removed by a microsurgical procedure and the resulting zygotes will be cultured to the blastocyst stage when the inner cell mass can be used for establishing human embryonic stem cell lines. Once the technique is established, the scientists will have a valuable resource in using the ESC lines for therapeutic applications and regenerative medicine. The most important aspect of this work is to further study the biology and differentiation of human embryonic stem cells. To date, none of the fertility clinics in the Northern California reserves or freezes these tripronuclear zygotes for research studies. Hence, there is an urgent need to bring attention to this unique resource in the field of human embryonic stem cells.
Statement of Benefit to California: 
Summary of benefit to California Obtaining human embryonic stem cell lines is the prerequisite for developing therapeutic approaches. The proposed research is to use the eggs with two sperm inside that are routinely discarded in the in vitro fertilization laboratory. There is evidence that these eggs could develop normally after removing the extra sperm. It is a valuable resource to generate human embryonic stem cell lines without ethical concern of using human embryos.
Progress Report: 
  • A main goal of research in our laboratory is to identify strategies to promote neural repair in spinal cord injury and related neurological conditions. On the one hand, we have been using mouse models of spinal cord injury to study a long-standing puzzle in the field, namely, why axons, the fibers that connect nerve cells, do not regenerate after injury to the brain and the spinal cord. On the other hand, relevant to this CIRM SEED grant, we have started to explore the developmental and therapeutic potential of human embryonic stem cells (hESCs) for neural repair. We do this by first developing a method to genetically manipulate a HUES line of hESCs. The advent of hESCs has offered enormous potential for regenerative medicine and for basic understanding of human biology. To attain the full potential of hESCs as a tool both for therapeutic development and for basic research, we need to greatly enhance and expand our ability to genetically manipulate hESCs. A major goal for our SEED grant-sponsored research is to establish methods to genetically manipulate the HUES series of hESC lines, which are gaining wide utility in the research community due to the advantages on their growth characteristics over previously developed hESC lines. The first gene that we targeted in HUES cells, Fezf2, is critical for the development of the corticospinal tract, which plays important roles in fine motor control in humans and hence represents an important target for recovery and repair after spinal cord injury. By introducing a fluorescent reporter to the Fezf2 locus, we are now able to monitor the differentiation of hESCs into Fezf2-expressing neuronal lineages. This work has been published. A second goal is to start to explore the developmental and therapeutic potential of these cells and cells that derived from these cells in the brain and spinal cord. We are currently utilizing the cell line genetically engineered above to develop an efficient method to differentiate HUES cells into subcerebral neurons. Results so far have been encouraging. Efforts are also underway to overexpress Fezf2 as a complementary approach to drive the differentiation of HUES cells into specific neuronal types. Together, these studies will lay down the foundation for therapeutic development with HUES cells and their more differentiated derivatives for neurological disorders including spinal cord injury where neural regeneration can be beneficial. The CIRM SEED grant has allowed us to pursue a new, exciting path of research that we would have not pursued had we not been awarded the grant. Furthermore, the CIRM funded research has opened a new window of opportunity for us to explore genetic engineering of hESCs to model human neurological conditions in future.

© 2013 California Institute for Regenerative Medicine