Stem cell therapy on hypertension: A potential source and role of nNOS in brainstem

Funding Type: 
SEED Grant
Grant Number: 
RS1-00416
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
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.
Progress Report: 
  • The original goal of this project was to generate oocytes (eggs) from human embryonic stem (hES) cells in cell culture dishes in the laboratory. Such oocytes could be of use as vehicles to reprogram the DNA from cells of patients with life-threatening or debilitating conditions, thereby allowing generation of new lines of hES cells that are immune matched to the patient. The paucity of donated human oocytes precludes research using such material, and production of human oocytes from hES cells in the laboratory would in theory provide a limitless source of material.
  • Since our last progress report another CIRM-funded group, Dr. Renee Reijo Pera’s lab at Stanford University, has published exciting results demonstrating successful production of primordial oocytes from mouse ES (mES) and human ES (hES) cells. Consequently, during the remaining period of the award we propose to use the Reijo Pera methods for production of female germ line cells in our lab using H9 and HUES-9 female hES cells. After accomplishing this, we will introduce human mtDNA containing mutations that cause either a severe or mild reduction in oxidative phosphorylation (energy) production into H9 and HUES-9 hES cells and investigate the impact of the different mtDNA mutations on the ability of hES cells to form cells with characteristics of PGCs, then primordial oocytes in vitro and in vivo. An important related goal of this research is to investigate whether development of oocytes from ES cells could be used as a method to remove deleterious mtDNA mutations from the hES cell population, thereby improving the utility and possibly safety of derived cell types for therapeutic purposes.
  • During this reporting period we have focused on our approved revised research plan. We continued our efforts to generate embryonic female germ cells from human embryonic stem (hES) cells in vitro using methods reported in the literature at the end of 2009. Our revised plan included the new goal of using in vitro developed female embryonic germ cells (oocytes) as a resource to investigate how mitochondrial genomic DNA containing deleterious mutations is segregated during female germ cell development. As well as providing novel information about the biology of germ cell development, this research may provide important information relevant to development of safe methods for therapeutic cloning. We began by using two different female hES cell lines (HUES-6 and H9) to investigate whether we could use the reported methods to develop female germ cells from hES in our lab. A third female hES cell line (HUES-9) we originally intended to use was found to have a high propensity to gain an extra chromosome (become aneuploid) and was therefore not used. Despite following the reported methods that demonstrated in vitro differentiation of hES cells into female germ cells, we were unable to reproduce the previously reported results in our own lab. The reasons for this are currently unclear but may involve subtle, but important, differences in the methodology or materials we used.

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