Ovol genes and hES differentiation into hair-producing cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00377
ICOC Funds Committed: 
$0
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Public Abstract: 
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.
Progress Report: 
  • We have shown that fetal human central nervous system derived stem cells (HuCNS-SC) transplanted into a mouse model of spinal cord injury (SCI) improve behavioral recovery. Transplanted human cells differentiated into myelinating oligodendrocytes and synapse forming neurons. These data suggest that efficacy is dependent upon successful cell engraftment and appropriate cell fate. The strain of mice (NOD-scid mice) are immunodeficient, which allows transplanted human cell populations to engraft and promote behavioral recovery in the absence of confounds due to a rejection response and allows us to avoid using immunosuppressant drugs. Clinically, however, it is clear that transplantation of therapeutic human cell populations will require administration of immunosuppressants (IS) such as CsA, FK506, or Rapamycin. These immunosuppressants work by altering signaling pathways which are also present within stem cells. Hence, in addition to promoting engraftment, IS have the potential to affect stem cell proliferation and/or differentiation. In Aim 1A, we tested this hypothesis in a cell culture model and found that HuCNS-SC fate and proliferation were altered by exposure to different IS. CsA and FK506 decreased the number of astrocytes in culture compared to control conditions, while Rapamyin increased the number of astrocytes. All three IS increased the number of ß-tubulin III positive neuron-like cells.
  • In Aim 1B, we tested whether cells of the inflammatory system (neutrophils and macrophages) could also directly influence stem cell proliferation and fate. To test this possibility, we exposed either fetal or embryonic neural stem cells to cell culture media from co-cultures of neutrophils or macrophages. We found that neutrophil-mediated release of inflammatory proteins promotes astrocyte differentiation of fetal derived neural stem cells but not embryonic derived neural stem cells. One way inflammatory cells might be working is via oxidative stress (e.g. hydrogen peroxide). Interestingly, excess hydrogen peroxide promoted more extensive cell death of embryonic derived versus fetal fetal derived neural stem cells, suggesting an intrinsic difference in the vulnerably of these two cell populations to oxidative stress. Conditioned media from neutrophils was found to reduce proliferation in fetal neural stem cells but not embryonic derived neural stem cells. In addition, we found neutrophil conditioned media promotes human fetal NSC astrocytic fate and migration towards sites of injury epicenter in an animal model of spinal cord injury; followup cell culture experiments enabled us to determine that neutrophil synthesized complement proteins were having a direct effect on stem cell fate and migration, resulting in a patent filing. These data demonstrate that fetal NSCs and ES-NSCs are very different by nature and nurture.
  • In Aim 2, we evaluated the hypothesis that IS could alter stem cell proliferation and/or fate in vivo, independent of rejection from the recipient’s immune system. HuCNS-SC were transplanted into NOD-scid mice, which have no immune system and hence cannot mount an immune response to the foreign cells. These animals received different immunosuppressants (CsA, FK506, Rapamycin, or vehicle) daily after transplantation until sacrifice 13 weeks later to determine if the total number of surviving human cells, or the end cell fate of the transplanted cells would be altered due to exposure to IS drugs compared to the vehicle control group. Behavioral recovery was assessed via open-field walking assessment, horizontal ladder beam testing, and video based “CatWalk” gait analysis. IS administration did not affect behavioral recovery by any of these measures compared to HuCNS-SC transplanted animals that received vehicle as an IS. Spinal cords were dissected, sectioned, and immunostained using human-specific markers in conjunction with cell lineage/fate and proliferation markers. Cell engraftment, proliferation, and fate were quantified using unbiased methods. The average number of engrafted human cells in uninjured animals was 319,700 vs 214,900 in vehicle treated injured controls. Human cell engraftment in any IS group was not significantly different than vehicle injured controls. Interestingly, 67% of human cells differentiated into Olig2+ oligodendrocyte-like cells in the uninjured controls, while 45% were Olig2 positive in vehicle treated injured controls. IS treatment did not alter Olig2 cell numbers in injured animals. 9% of human cells differentiated into GFAP positive astrocyte-like cells in the uninjured controls, compared with 9% in vehicle treated injured controls. IS treatment did not alter GFAP cell numbers in injured animals. Quantification of proliferation and other lineage markers is ongoing. The important finding thus far is that when administered to whole animals with a human stem cell transplant, a range of immunosuppressant drugs does not appear to significantly alter stem cell fate.

© 2013 California Institute for Regenerative Medicine