The overall goals of this application are to examine the similarities of human embryonic stem (hES) cell-derived neural stem cells (NSC) with human amniotic epithelial (AE) cells and to develop an “alternative” stem cell therapy to treat hypertension. Hypertension or high blood pressure is a major public health problem with serious medical and financial consequences. Nitric oxide (NO) is an essential molecule in the nucleus tractus solitarius (NTS) in brain which regulates cardiovascular functions and decrease blood pressure. We have recently shown that neuronal nitric oxide synthase (nNOS, an enzyme for synthesizing NO) is decreased in the NTS region of Dahl salt-hypertension-sensitive (DS) rats with an elevated blood pressure. Recent studies have demonstrated that NSC can differentiate into neurons, replace damaged neurons, and express nNOS. Human AE cells express markers for pluripotent stem cells, and synthesize/release neurotransmitters. Our preliminary results show that cultured human AE cells and NSC express both embryonic & neural stem cell markers. Interestingly, nNOS is consistently expressed in cultured NSC and human AE cells. These results suggest that human AE cells may potentially function as NSC after they are differentiated into NTS neurons. We hypothesize that human AE and NSC cells have similar characteristics, and transplantation of human AE cells to the NTS region in the hypertensive animals will control their hypertension. In view of the great therapeutic potential of stem cell therapy and importance of nNOS-NO expression in hypertension, our major aims are: 1) Examine whether neuronal, embryonic stem cell markers, and nNOS are expressed in cultured human AE cells compared to NSC; 2) Determine expressions of nNOS and stem cell markers in the NTS is enhanced, and high blood pressure is reduced by transplantation of human AE cells vs. NSC into the NTS region in DS rats with salt load; and 3) Investigate whether in-utero transplantation of human AE cells vs. NSC during pregnancy induces nNOS expression in the NTS and controls hypertension in DS offspring with salt load. Cells will be labeled with tracking red dye or adenovirus, and then injected into the NTS in young DS rats or pregnant DS rats’ uteri. Blood pressure and heart rate will be monitored, and nNOS mRNA and protein expressions in the brain nuclei will be analyzed to test the hypotheses in genetically altered DS rats. The discoveries will yield new insights regarding stem cell therapy for nNOS induction in the NTS to prevent and control genetic hypertension. Utilizing human AE cells serve as an easy, accessible and high yield cell source in which cell recovery does not provoke serious ethical debate for stem cell therapy. These studies should benefit the public health by advancing our understanding of how stem cells are involved in nervous system for prevention and treatment of hypertension.
Statement of Benefit to California:
Hypertension is a major public health problem with serious medical and financial consequences. The mechanisms of pathogenic hypertension and effective strategies for prevention and treatment of hypertension remain unclear. Several postulates have been proposed to explain an imbalance and an abnormal modulation of the sympathetic system, which include genetic, stress, cultural, and environmental factors. The nucleus tractus solitarius (NTS) in the brainstem is the principal sensory nucleus for the central regulation of cardiovascular function through central sympathetic pathways. Recent studies show that nitric oxide (NO) in the NTS plays an important role in the central inhibition of sympathetic tone and decreases arterial blood pressure. The proposed experiments will determine whether utero or local transfer of human amniotic epithelial (AE) cells and neural stem cells (NSC) derived from human embryonic stem (hES) cell lines induce neuronal nitric oxide synthase (nNOS) in the brainstem as a method for hypertension control. In view of the critical importance of NO on sympathetic and cardiovascular regulation, it is necessary to address the mechanisms of stem cell on NO-mediated cardiovascular regulation and develop a stem cell therapy to prevent and treat patients with hypertension and cardiovascular diseases. The overall goals of this application are to examine the similarities of human AE cells with NSC derived from hES cell lines and to develop a new source of stem cell therapy to control genetically-altered hypertension. The proposed experiments will compare the neural embryonic stem cell characteristics of cultured human AE cells with NSC, and test the therapeutic effects of NSC and AC cells on hypertension control following utero or local transplantations. It is no doubt that stem cells hold great therapeutic potential aimed at maintaining, restoring, or enhancing tissue and organ function, which is intended to treat a number of disorders. Although the State of California is ineligible to study embryonic stem cells, the use of these cells raises several ethical concerns. Moreover, stem cell-based regenerative medicine seems to be hindered by the high rate of tumor induction and immunological refection after transplantation. It is necessary to identify a source of stem cells that is safe and easily accessible, provides a high cell yield and for cell recovery does not provoke serious ethical debate. Human AE cells from the placental tissue display an easily accessible, safe and high cell yield source, which its use raises no ethical controversy, and its procurement is safe and non-restricted in availability. These studies should benefit the public health and California citizens by advancing our understanding of stem cell mechanisms and functions of human AE cells involved in NOergic system for pathogenic and treatment of hypertension.
SYNOPSIS: Nitric oxide (NO) in the nucleus tractus solitarius (NTS) plays an important role in decreasing blood pressure (BP). The hypotheses of this proposal are: both human amnion epithelial cells (AECs) and hESC-derived neural stem cells (NSCs) possess NSC characteristics and express nNOS; transplantation of hAECs and NSCs to the NTS and/or utero will increase nNOS expression and decrease BP. In Aim I, the PI will examine whether nNOS, eNOS, NSC markers, neuronal markers, and ESC markers are expressed in cultured hAE cells as well as hESC-derived NSCs. In Aim II, the PI will determine whether nNOS expression in the NTS is enhanced and BP reduced by local transplantation of hAE cells or NSCs in DS rats with a salt load. In Aim III, the PI will investigate whether utero transplantation of hAECs or NSCs during pregnancy induces nNOS expression in the NTS and controls BP in these rats. The long range goals of this project are to investigate NSC generation of nNOS in the brainstem, and to develop a hAEC BP treatment. INNOVATION AND SIGNIFICANCE: Hypertension is clearly a major health problem at present with suboptimal treatments. The idea of raising levels of NOS in the brainstem as a treatment of hypertension is novel. AEs cells are early accessible, have few ethical concerns about their use, lack MHC, and may be a good alternative to hESCs with respect to certain treatments. Their proposed use as a substitute for hESCs is an innovative one. STRENGTHS: A few strengths were noted for this proposal. First, the subject of this proposal, the treatment of hypertension, is an important one and the PI has creative ideas with respect to the control of blood pressure by transplantation of hNSCs or hAECs. Second, the in vitro characterization of hAECs followed by animal transplantation studies is a logical and powerful test of the PI’s hypotheses. Third, the PI is an expert on the relationship between NO and cardiovascular responses. Finally, the collaboration with Yi Sun is a valuable one. WEAKNESSES: The reviewers noted several weaknesses of this porposal. First, this project is unlikely to be completed within 2 years. Second, cells will not be developed within the PI's lab but instead the hESC-derived neural stem cells are being aquired from a collaborator, Dr. Yu Sun, at UCLA and the amniotic stem cells will be purchased from a commercial laboratory, ScienCell. Third, the hES-derived neural stem cell studies appear to be an add-on to an on-going study employing amniotic stem cells. It is not clear why two cell types are needed for these aims at this stage of the work. Fourth, the proposal is generally a bit difficult to follow. For example, one is not quite certain whether the main thrust of the proposal is to demonstrate that increasing levels of NOS is effective in treating hypertension-induced rats or to characterize hAECs and show that they are in many ways comparable to NSCs; this aspect of the proposal needs some more clarification. Fifth, the Western blot provided in Fig. 2 is of interest, however, it would have been valuable for the PI to have shown or noted that an equal amount of protein was applied to the lanes that were compared. Also, how does the PI know that only the NTS region was actually obtained for the analysis? Will faulty identification of different brain regions for the laser dissection work proposed in Specific Aim 3 cause difficulty in interpretation – and how can this be avoided? Sixth, the PI states that “transplantation of human AE cells...can be differentiated into mature NTS neurons.” What is the evidence that this can occur? How would the PI go about carrying out this differentiation? How long are cells kept in culture? How many of the cells become differentiated over what period of time? How pure are the cells used for transplantation? Seventh, more information should be provided about the characteristics of hES cell-derived NSCs that will be used. Finally, the PI generally does not adequately address possible pitfalls to the experiments and alternative approaches that may be taken. DISCUSSION: There was no further discussion following the reviewers' comments.