Comprehensive study of the osteogenic potential of human embryonic stem cells - are they equivalent to liposuctioned fat and bone marrow derived stem cells?
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
SYNOPSIS: The PI, a senior academic plastic surgeon, aims to build on previous experience in creating bone-scaffold structures that are used experimentally for repairing cranial defects from disease or trauma. In the past the PI has used mesenchymal stem cell lines, primary human bone marrow-derived stem cells, and stem cells isolated from liposuction specimens for seeding the scaffolds. The overall goal of the SEED grant proposal is to compare hESCs as a source for bone on these scaffolds to the other stem cell types. The specific aims are (i) to determine the optimal doses of dexamethasone, retinoic acid and vitamin D that promote osteogenic differentiation in cultured hESCs; (ii) Compare hESCs on 3-D scaffolds for ability to form bone compared to the other stem cell types (Note: Prelim data suggest that the 3-D scaffolds downregulate bone formation in the other stem cells compared to 2-D culture); and (iii) to characterize gene expression changes in hESCs on 3-D scaffolds compared to 2-D controls. INNOVATION AND SIGNIFICANCE: As the population ages, problems like osteoporosis become more prevalent. Developing stem cell -based or -derived therapies to repair bone defects are therefore significant. Studying mesenchyme formation and subsequent osteoprogenitor and osteocyte formation from hESCs might yield cells to gain novel insights in the earliest developmental steps and processes in humans. hESCs have not been used as sources of cells to populate bone scaffolds. Because of the immediate translatability of these kinds of constructs for repair of cranial trauma, the work is innovative. (Small point: The PI does not acknowledge the fact that fat-derived stem cells may be ‘better’ as a source for the proposed constructs, since they can be harvested autologously in most Americans. But this point also highlights the fact that the case for hESCs as a potentially superior source of cells compared to those the PI has worked with is not strongly made in the proposal.) STRENGTHS: The major strength of the work is the historic experience of the PI with bone differentiation, use of scaffolds in osteoprogenitor generation, and a clinically relevant animal model for testing engineered bone constructs that use a variety of stem cell sources. Another strong point is the comparative nature of the study where two adult sources of precursors will be compared with hESCs for the efficiency of bone formation. WEAKNESSES: The PI did not integrate some of the recent literature into the proposal. The major hitch in the research plan may be that hESCs given the same signals (dex, RA, vitD) as other stem cells may not be as likely to differentiate toward bone, without an intermediate mesenchymal stem cell step. So the timing of the differentiation should be worked out (and likely more parameters played with) in the hESCs before anything else is done. There are good suggestions in the literature of manipulations likely to increase bone differentiation from hESCs. Although the PI proposes to test a variety of doses of the three inductive factors, the timing of these additives may also be important in the hESCs. So it is likely to take a bit more time to coax these cells with any real efficiency to bone. Furthermore, the investigators will not exploit the strength of hESCs, namely to aid in understanding the molecular mechanisms underlying bone formation from ESCs to mesoderm, mesenchyme and then osteprogenitor generation. The grant application would be stronger if the study on hESCs was emphasized more, since the large historical experience of the lab with other stem cells may not have to be repeated for all the comparisons. If hESC bone induction were a larger focus of the proposal, the success of coaxing these cells to bone would be increased. On another point, the structural testing of the quality of bone would also be a good addition, or at least use in the animal model (which is not mentioned in the grant application except as preliminary data). The PI has checked off that human oocytes will be used, but there is no indication of this in the grant application. The use of in vitro embryos and derivation of new lines are also indicated but not described. The PI mentions use only of H1 and H9 lines. Finally, no methods are proposed to assess how teratoma formation will be avoided if ESC-derived cells were to be used in the clinic. DISCUSSION: This proposal uses a "neat" cranial defect model, and a scaffold that can repair defects is intriguing and immediately transferable. The main concern is that the simple addition of dexamethasone and other agents may not drive hESCs straight to bone. There are likely intermediate differentiation steps needed.