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RC1-00340-1: Engineering human embryonic stem cells into ectodermal organs

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Recent progress in human embryonic stem cells (hES) offers tremendous hope in regenerative medicine. hES have been guided to replace several key cell types for some devastating diseases. However, the potential of hES in skin and associated ectodermal appendages, the organ that suffers from constant wear and tear, has not been widely explored to date. The overall goal of this proposal is 1) to understand how human embryonic stem cells (hES) can be guided to form ectodermal organs, including epithelial components (hair, teeth, skin, glands, etc.) and mesenchymal tissues (bone and periodontium), and 2) to explore the potential use of hES in tissue regeneration to repair or replace orofacial epithelial and mesenchymal defects. These defects often result from burns, accident injuries, congenital anomalies, genetic diseases, cancers, infectious diseases, etc. There is also a great demand for tooth replacement and bioengineered hair follicles.

One of the major problems for regenerative medicine is the lack of sufficient numbers of precursor cells. We expect that advances in the field will help generate sufficient numbers of precursor cells from hES. In this proposal, we will focus to develop and establish novel procedures so these hES cells or derivatives can be guided to form the epithelial component of ectodermal organs and the associated mesenchymal structures. To this end we will mimic different microenvironments and do experiments to identify the appropriate key ingredients of molecular signals that specify hES to build ectodermal organs and associated structures.

If the work proposed here is accomplished, it would significantly expand the therapeutic potential of hES. Furthermore, hES mediated ectodermal organ formation will be a major novel scientific progress. As we know, bone marrow transplantation is already a mature technology. This is because the derived blood cells are released to the blood stream without the need to build a complex three-dimensional architecture. Ectodermal organs, endodermal organs (e.g., liver) and neural tissues (neural circuits, but not including dopaminergic neurons for Parkinsons disease, in which only the released neurotransmitters are required) all require organized arrangements of cells/tissues for proper function. Learning how to build ectodermal organs will have implications beyond ectodermal organs. This is also more than just making a flat epidermis, which currently is already achieved by foreskin keratinocyte transplantation.

The head is the body part in which ectodermal organs and associated structures are most important as well as most subject to injury. Therefore, we will have ectodermal organs in the head and orofacial regions as our priority goals. We also have been interacting with Dr. Garner, Burn Unit Chief in USC, and will explore future clinical possibilities after we accomplish the work proposed here.

Statement of Benefit to California (provided by applicant)

Recent progress in human embryonic stem cells (hES) offers tremendous hope in regenerative medicine. hES have been guided to replace several key cell types for some devastating diseases. However, the potential of hES in skin and associated ectodermal appendages, the organ that suffers from constant wear and tear, has not been widely explored to date. The overall goal of this proposal is 1) to understand how human embryonic stem cells (hES) can be guided to form ectodermal organs, including epithelial components (hair, teeth, skin, glands, etc.) and mesenchymal tissues (bone and periodontium), and 2) to explore the potential use of hES in tissue regeneration to repair or replace orofacial epithelial and mesenchymal defects. These defects often result from burns, accident injuries, congenital anomalies, genetic diseases, cancers, infectious diseases, etc. There is also a great demand for tooth replacement and bioengineered hair follicles.

In this proposal, we will focus to develop and establish novel procedures so hES cells or their derivatives can be guided to form the epithelial component of ectodermal organs and the associated mesenchymal structures. To this end we will mimic different microenvironments and do experiments to identify the key ingredients in molecular signals that specify hES to build ectodermal organs and associated structures.

The head is the body part in which ectodermal organs and associated structures are most important as well as most subject to injury. Therefore, ectodermal organs in the head and orofacial regions will be our priority targets. In California, people live an outdoor lifestyle and are subject to more UV irradiation and associated diseases. Californians are also more conscious of their appearance and having imperfect ectodermal organs (e.g., alopecia) can cause psychological distress. With CIRM funding, we hope to develop remedies for these conditions. To do this, we wish to find the best possible hES lines for this purpose. Current Federally approved hES have some problems (see text) and we should not be limited to them.

If we can produce ectodermal organs using methods as routine as bone marrow transplantation (which works easier because it does not require the complex three dimensional structures found in other organ systems), the need for replacement / regenerative ectodermal organ therapy will be huge. It will not only benefit Californians but also the California biotechnology industry. We also have been interacting with Dr. Garner, Burn Unit Chief, and Dr. Woodley, Chair of Department of Dermatology in USC. We will explore future clinical possibilities after we accomplish the work proposed here. This field is ready. With CIRM support, we will be able to delve into this novel, risky but high return endeavor.

Review

SYNOPSIS: This application proposes to engineer human Embryonic Stem Cells (hESCs) into non-neural ectodermal organs (hairs, tooth, skin, glands) which are subject to wear and injury. The Principal Investigator (PI) hypothesizes it should be possible to mimic micro-environmental signals to direct hESCs to non-neural ectodermal lineages and then to differentiate ectodermal organs. Since ectodermal organs are integrated with specialized connective tissues, the second part focuses on guiding hESCs to become mesenchymal stem cells and to give rise to bone marrow in orol-facial bones, periodontal ligaments, dental pulp and other associated essential mesenchymal structures.

IMPACT & SIGNIFICANCE: The focus of this application is to develop the epithelial component of ectodermal organs and their associated mesenchymal structures from hESCs. The applicant aims to understand how hESCs can be guided to form these organs and to explore the use of hESCs in tissue repair or replacement with particular emphasis on orol-facial epithelial and mesenchymal cell defects such as those resulting from burns, accident injuries, congenital abnormalities, cancers, or infectious diseases. Thus while much of the present interest relates to burn injuries, additional applications might present themselves with enhanced ability to isolate and differentiate cells.

The ability to repair ectodermal organs may be of significance in the future and the prospect of engineering complex tissues from stem cells is one of the most exciting applications of hESC research. The reproducible differentiation to each cell type, as proposed in this application, is a required intermediate goal.

QUALITY OF THE RESEARCH PLAN: The focus of this proposal is on the craniofacial region as it is most relevant clinically. Ectodermal organs (skin, hair, teeth, sweat glands, etc) are products of epithelial-mesenchymal interactions and share signaling mechanisms. The applicants hypothesize that it would be possible to identify and mimic micro-environmental signals to make hESCs become non-neural ectodermal stem cell lineages and eventually different ectodermal organs.

The first Aim focuses on the epithelial component, exploring ways to guide hESCs (inducers) to “products” (cells) that will be transplanted to SCID mice to evaluate tissue and organ-forming potential and clinical potential for transplantation. To that end they propose to start with cell lines from their collaborator. However, they will also evaluate differentiation potentials of non-NIH approved hESC lines such as those brought by another collaborator. They intend to use a variety of culture methods with which the PI’s laboratory has extensive experience to determine whether hESC-derived epithelial cells will be able to form hairs or teeth. Differentiation will be tested by tissue recombination. Then they will assess standard markers to analyze for differentiation, measuring changes in molecular expression during this progression of hESCs to epithelial organs. They will perform microarray experiments in temporal fashion, hoping to identify major regulators; and test by siRNA. They hope to learn how much the in vitro development compares to the events that occur in vivo. They then will test hESC-derived ectodermal organs for in vivo function using several established assays.

A second component of their work is based on the need to mold hESCs into complex 3-D structures. This will involve hESC differentiation into mesenchymal stem cell populations including marrow, dental pulp, periodontal ligament, etc. The PI will follow described protocols using various factors and also will use surface markers to isolate cell populations. The PI will evaluate biomarker expression, perform over-expression experiments, and compare efficacy of mesenchymal stem cells from hESC and bone marrow, eventually to be tested in vivo. The in vivo experiments will use SCID mice and will include attempts at repairing defects. Overall, these studies have been logically laid out.

The PI has studied feather and limb development, and enlists the assistance of a surgeon, who is skilled in micromanipulation. A collaborator of the PI has worked out conditions for clonal growth and population expansion of human keratinocytes for 40-60 doublings and will provide cells and advice as needed. Also, another collaborator and colleagues showed that cells expressing markers consistent with the keratinocyte lineage can be isolated from embryoid bodies and teratomas formed in SCID mice. These cells have limited proliferative potential but can be immortalized. The cells selected also express markers of stratified epithelial cells. Also the cells form multilayered epithelia. The PI hypothesizes that these cells represent incomplete state of development of stratified epithelial lineage and may be candidates for susceptibility to “instruction” by embryonic mesenchyme to form hair or tooth structures.

The plan to observe gene expression changes during differentiation to ectodermal and mesodermal lineages is fairly straightforward and standard. However, the plan for differentiation of hESC along ectodermal lineages is ambitious compared with the relatively small amount of preliminary data supporting the plan. It is very likely, based on the observations others have made of similar co-cultures attempted for maintenance of hESC, that a broad range of differentiation products will be obtained, not simply ectoderm. This is also true for over-expression of specific factors, which have roles at many levels of development. In addition, the low proliferative capacity of hESC progeny do not support the likely success of the in vivo studies. The second aim, which proposes to differentiate cells of mesenchymal lineages, has similar issues. The study proposing to induce mesenchymal differentiation is without sufficient preliminary data to suggest that this plan will yield mesenchymal cells from hESC. Bone marrow stroma has in fact been proposed as a good source of feeders to prevent hESC differentiation. More problematic is the lack of a plan to identify the hESC-derived cells from the co-cultured mesenchymal cells. If mesenchymal differentiation could be identified and cells isolated from the mix of cell types, the plan to over-express genes found to be influential in mesenchymal stem cells may be fruitful. However, plans to make complex transplants are somewhat premature based on the lack of success using similar cell types from primary tissues, especially since the plan was lacking a rationale for why hESC would yield better results. Data from a collaborator’s lab do not support this notion.

STRENGTHS: The PI’s group has been at the forefront of ectodermal organ biology. The PI and his/her collaborator have experience in stem cell biology as evidenced by a recent Nature paper on mapping of stem cell activities in the feather follicle. Further, they have shown in previous studies that human bone marrow mesenchymal cells are capable of forming bone/marrow and dentin/pulp-like structures when transplanted into mice using a carrier. They show some impressive examples of preliminary studies.

The PI has a great deal of experience with feather development, which is valuable in understanding ecotermal developmental patterns. Collaborations with the a stem cell core facility at his/her instituion and with a collaborator will provide needed expertise in using hESCs. Input from another collaborator is considered a strength as well.

WEAKNESSES: Reviewers agreed that the plan for transplanting the tissues is premature, without the basic studies to support the differentiation strategies having been worked out already. In addition, no rationale was provided for expecting that hESCs will succeed in vivo where primary cells have not, reducing enthusiasm for the proposal.

A reviewer pointed out that while in vivo testing in an immunodeficient mouse model is important to document the function of both epithelial and mesenchymal stem cells, these studies bear no resemblance to an eventual clinical application. In the latter setting, graft rejection can safely be assumed owing to incompatibilities for both HLA and non-HLA antigens. It is unclear how the applicants intend to get around this problem.

A reviewer suggests that the PI focus more on the quality of starting cells and conditions for their isolation etc. since in his/her view, the starting cells and methods for differentiation are not particularly imaginative. The PI seems to assume that the cells that were selected to be used represent the best place to start, which is not necessarily the case. Preliminary studies to give some indication as to whether or not the differentiation schemes will yield the cell types required for the studies proposed is considered by the reviewers to be key. In addition, reasonable plans for distinguishing the ectodermal cells from the mix of differentiated cell types, as well as the hESC-derived mesenchymal cell types from the co-cultured primary mesenchymal cells, is needed for the success of aim 2.

DISCUSSION: The PI has extensive experience in avian systems, and one reviewer views as a particular strength the track record of the PI on the feather follicle, considering the Nature paper to be elegant work. This reviewer has confidence that the PI can carry out the proposed studies. The PI does not have much experience with hESCs, but the collaboration with with an expert in this area is good. However, reviewers generally agree that there is a lack of rationale and preliminary data for this work. The idea of building complex organs is considered by the discussants interesting, exciting, and important, but it is too early to consider these experiments. One reviewer is particularly concerned that the starting cells are poorly characterized and is not convinced that their culture conditions will give rise to the desired differentiated cells; it has not been shown that culturing ESC with another cell type will make them like that differentiated cell type. In fact, it has been shown that culturing ESC with bone marrow stromal cells prevents their differentiation; so why does this applicant expect a different result? Also, there is no plan to eliminate the endpoint cells from the mesenchymal co-cultured cells. Another main weakness is the in vivo testing of functionality as no attempt is made to deal with potential graft rejection problems.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None