Each year in America, there are 700,000 emergency visits and 45,000 hospitalizations for burn treatments, and 4,500 deaths due to fire and burns (http://www.ameriburn.org/resources_factsheet.php). Since twenty years ago, the use of artificial skin generated from human skin cells has tremendously improved the survival rate of severely burned patients. However, this technology suffers a major drawback: the artificial skin lacks the various structures that are part of the normal skin, such as hair follicles, sweat glands, and cells that make pigments or fight pathogens. Embryonic stem cells are cells that are isolated from early embryos and have the potential to make all kinds of cells in the human body. Conceivably, they hold great promises to help generate a new kind of artificial skin containing all residential structures and cell types that would function just like real skin.
Hair follicles play many important roles in skin – they provide a passage for perspiration, are the source of cells that are used for natural wound repair, and the house of other important cell types. We therefore will focus our effort to first use hES cells to make hair follicle-containing skin on Petri dishes. Theoretically, a hES cell has to make three important, sequential fate choices during its path to become a hair follicle cell. First, it has to choose a fate that has the potential to be part of the nervous system or part of skin. After this, it has to then choose a skin fate over a neural fate. Last, it has to choose to become a hair follicle cell instead of just the skin between follicles. We will experimentally manipulate these fate choices by adding or removing key regulatory proteins (such as growth factors) that are known to be involved in these decisions based on studies in model systems such as mice. Clearly, this is a long-term process: we not only have to find conditions that would allow the generation of some hair-producing cells, but also have to optimize the conditions so that we can generate many of such cells and have them distribute in a pattern that resembles the distribution of hair follicles in normal skin. Our goal in the current grant application is to be able to generate some hair-producing cells from hES cells. This, if successful, will lay the ground for future work from our as well as other laboratories. Ultimately, we may be able to generate hair follicle-containing skin to be used for transplantation not only onto burn patients, but also onto healthy people with baldness.
Statement of Benefit to California:
Wildfire has been an unfortunately frequent presence in the State of California. As the state continues to grow, more and more people live in forest areas, facing a high risk of physical (and property) damage from wildfire. For example, a total of 3500 houses were burned in the Oakland/Berkeley fire in 1988 and Painted Cave fire in 1990. This, together with other causes of fire and burn such as use of defective product and accidents, leaves Californian citizens suffering from a high incidence of burns and injuries. The studies we propose in this application may yield findings that will directly benefit the health and welfare of burn patients in California. Furthermore, these findings may also be used to help establish industrial entities in the State of California that perform research, development, and marketing of new therapeutic products. This will create new job opportunities for California citizens and increase revenues for the State of California.
SYNOPSIS: The goal is to develop conditions under which hESCs can be directed to differentiate into human skin, complete with all the functions of normal skin, as a replacement tissue for burn patients. Aim 1 extends preliminary results in mice to determine the role of the zinc finger transcription factors Ovol1 and Ovol2 in directing ESC fate toward epidermal ectoderm. Generation of Ovol2-overexpressing cells will test whether Ovol 2 is required for maintaining pluripotency and for epidermal commitment. Generation of cells with decreased Ovol1 expression will test the role of Ovol1 in BMP4-induced trophectoderm differentiation. Aim 2 seeks to develop a protocol to differentiate hESCs into hair follicles. hES cell lines expressing EGFP under the direction of a hair-specific promoter will be used to monitor hair cell specification. The Ovol2-overexpressing cells of Aim1 will be tested to determine whether increased Ovol2 improves the frequency of epidermal/hair follicle differentiation.
SIGNIFICANCE AND INNOVATION: Current burn therapy, which uses self-renewing human keratinocytes for wound repair, cannot regenerate essential features of functional skin, such as sweat glands, hair follicles, sebaceous glands and does not establish resident populations of dendritic cells and melanocytes. With multipotent epidermal cells derived from human ES cells, it should be possible to regenerate skin with more normal function.
This application proposes to test the role of two transcription factors, Ovol1 and Ovol2 in hES cell differentiation into hair follicles. While these two genes are important in epidermal diffentiation, this seems to be a risky and untenable hypothesis. There is no evidence to support the notion that hair follicles can be selectively induced.
STRENGTHS: The PI brings extensive expertise on epidermal development, especially the nature and function of Ovol1 and Ovol2, two key regulators of ectoderm formation and differentiation and the focus of this proposal. An insightful experimental plan for Aim 2 to induce and monitor epidermal cell development in hES cell cultures based on understanding the developmental biology of skin and hair follicle formation, particularly the role of growth factors and morphogens.
The investigator has a number of interesting "sub-hypotheses" that could be honed down to one or two and focussed on more deliberately and supported with preliminary data.
The common expression pattern of Ovol2 and nanog could be potentially interesting or a useless association. Further functional/mechanistic data are needed to support the connections being tested in aim1.
WEAKNESSES: This proposal is to test two hypotheses based on limited evidence. First, Ovol2 is required for hESC pluripotency, because it is expressed in mouse and human ES cells and in mouse ICM. Second, Ovol1 is important for trophoectoderm differentiation, because its level increases with BMP4-induction of cultured human ES cells toward trophoectoderm. More preliminary evidence in support of these hypotheses is needed to enhance the value of testing them.
The experiment to over-express Ovol2 in human ES cells seeks to prove two contradictory outcomes – that high Ovol2 will increase both ES cell pluripotency and epidermal commitment. This may occur in vivo when these possible actions would be temporally separated, but constitutive over-expression is unlikely to be a useful test.
The investigator proposed too many ideas to be tested, with little experimental justitfication for any of these.
DISCUSSION: There was no further discussion following the reviewers' comments.