Comprehensive study of the osteogenic potential of human embryonic stem cells - are they equivalent to liposuctioned fat and bone marrow derived stem cells?

Funding Type: 
SEED Grant
Grant Number: 
RS1-00271
ICOC Funds Committed: 
$0
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Bony defects of the face, skull, or long bone may result from trauma, destruction by tumors, or congenital causes like cleft palate. These defects are encountered regularly by plastic surgeons and current methods of reconstruction primarily involve bone grafts harvested from another part of the body (autologous bone) and re-planted into the defect. However, the amount of donor bone is limited and the grafts are difficult to shape. In addition, autologous bone adds an additional operative procedure and can result in pain, hemorrhage, fracture, and nerve injury. To solve these problems, our laboratory has been developing a bone graft substitute since 1990. In other words, we are trying to grow new bone in a pre-determined three-dimensional shape. Growing new bone instead of transferring it from one part of the body to another would dramatically reduce operative time, hospitalization, and morbidity. There are three ingredients necessary to grow new bone 1) a 3-D delivery scaffold 2) stem cells 3) factors that turn the stem cells into bone. A scaffold must be biocompatible and biodegradable and allow for cells to attach, multiply, and turn into bone. Our lab has been using a material called poly-lactide-co-glycolide (PLGA) to create scaffolds that degrade in the body into lactic and glycolic acid which are naturally found. PLGA is FDA approved and is currently used in dissolvable suture material. Osteoblasts, the cells that make bone, adhere to, multiply, and form bone on 3-D PLGA scaffolds. Another critical component of synthesizing bone is the stem cell source. Ideally, one could harvest stem cells from an individual, seed them onto a biodegradable scaffold of the necessary shape, turn the stem cells into bone, and implant the bone back into the same individual. We have used bone marrow stem cells (BMSC) grown on a 3-D PLGA culture to grow new bone in vitro (in cell culture) and in vivo (in a living organism) to heal cranial defects in rabbits. Liposuctioned fat cells have also been shown to form new bone in 3-D culture and in animal defects. There have been only a few studies investigating hESC’s ability to form bone on a 2-D plate or in a collagen gel, but no studies have looked at their ability to form bone on a 3-D scaffold. Our first goal is to seed hESCs on our 3-D PLGA scaffold and form bone. We will perform this experiment side by side with human liposuctioned fat and bone marrow derived stem cells for comparison. Our second goal is to supplement osteogenic media with different concentrations of Vitamin D and/or retinoic acid to determine an ideal growth media for maximal bone formation. Our third goal is to look at the specific mechanisms through which hESCs form bone and a blood supply. We will analyze the differences in gene expression of hESCs grown on 3-D culture and compare them with human fat and bone marrow derived stem cells. Understanding these mechanisms is critical to developing a bone graft.
Statement of Benefit to California: 
In 2001, there were 24,298 operations for craniofacial trauma in the United States, 227,500 births with craniofacial defects resulting in 37,732 operations to repair congenital craniofacial defects. It has been calculated that annually, approximately 3.6 million craniofacial cases were treated in the medical system [Snowden et al., 2003]. The rate per 100,000 of congenitial anomalies is 36.1, craniofacial trauma 119.5, and neoplasms 94 for an annual cost of approximately 23,206 million dollars[Snowden et al., 2003]. California is no exception to the types of trauma, congenital defects, or head and neck cancers that result in craniofacial skeleton defects. With the passing of the California helmet law, there is also decreased mortality of motorcyclists and bicyclists resulting in greater number survivors who might require repair of cranial and facial trauma defects. A bone graft substitute would have widespread use for the citizens of California. The development of a bone graft substitute would eliminate the pain and complications of graft harvesting, such as excessive blood loss, infection, and fracture. A bone graft substitute would significantly reduce operating time and hospital stay due to prolonged donor site pain and morbidity. Our purpose is to use an FDA approved biodegradeable and biocompatible 3-D scaffold seeded with stem cells exposed to osteo-inductive agents to grow new bone. Our study will significantly improve our knowledge about new bone formation, the osteogenic potential of different stem cell types, and likely result in a clinically viable and cost-effective bone graft substitute for use in the population of California
Progress Report: 
  • A promising approach to alleviating the symptoms of Parkinson's disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have been examining which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and have identified necessary cues. We have now extended these studies to determine that two of the molecules have dramitc effects on dopaminergic neurons made from human embryonic stem cellls and that at least in vitro, cytokines do not mask these effects. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.

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