The ability to propagate high quality human embryonic stem cells (hESCs) that are capable of giving rise to multiple cell types is essential in using hESCs to treat human diseases. Understanding the signaling pathways that control the maintenance of pluripotency and differentiation of hESCs is necessary for endowing such ability. The goal of this project is to determine whether HER2, a receptor tyrosine kinase, is essential in maintaining hESCs. HER2 is a multiple functional protein. De-regulation of HER2 expression or signaling is implicated in several human diseases. For example, over-expression of HER2 is found in 20-30% of breast cancer patients. Herceptin, a humanlized blocking monoclonal antibody, has been approved by the Food and Drug Agency to treat breast cancer patients. HER2 is required as an essential partner for the response of cells to multiple growth factors, including neuregulin (NRG). Mutation of the NRG1 gene is associated with Schizophrenia in several populations. Studies in mice suggest that HER2 may play a role in the etiology of Hirschsprung disease and in preventing dilated cardiomyopathy and muscular dystrophy. Interestingly, some breast cancer patients treated with Herceptin develop cardiac dysfunction, revealing multiple functions of HER2 and the complication of designing an ideal therapy. NRG1 has been shown to play a role in the formation of the conduction system (pacemaker) of the heart in mice. Improper processing of NRG1 may play a role in Alzheimer's Disease and neuropathy. We found that NRG1 plays a role in the recovery rate of rat neural stem cells. HER2 is highly expressed in undifferentiated hESCs. These data demonstrate that HER2 has pleitropic effects on multiple cell types and organs and suggest that HER2 is capable of integrating diverse signaling pathways that are essential in the control of maintenance and/or differentiation of hESCs where HER2 is highly expressed. Understanding whether and how HER2 regulates the maintenance and/or differentiation of hESCs may provide insight into harnessing the strategy to grow and use hESCs to treat human disease.
Statement of Benefit to California:
The ability to propagate high quality human embryonic stem cells (hESCs) that are capable of giving rise to multiple cell types is essential in using hESCs to treat human diseases. Understanding the signaling pathways that control the maintenance of pluripotency and differentiation of hESCs is necessary for endowing such ability. HER2 is a receptor tyrosine kinase and has been shown to play essential roles or is implicated in breast cancer, dilated cardiomyopathy, muscular dystrophy, heart pace maker, peripheral neuropathy, Schizophrenia and Alzheimer's Disease. For example, over-expression of HER2 is found in 20-30% of breast cancer patients. Herceptin, a humanlized blocking monoclonal antibody, has been approved by the Food and Drug Agency to treat breast cancer patients. Several lines of evidence suggest that HER2 is a multiple functional protein and may play essential roles in the maintenance and/or differentiation of hESCs. Understanding whether and how HER2 regulates the maintenance and differentiation of hESCs may provide insight into harnessing the strategy to grow and use high quality hESCs to treat human disease.
SYNOPSIS: The general goal of this project is to understand the signaling pathways that control the maintenance of human embryonic stem cells (hESC) in vitro. The experiments focus specifically on the signaling pathway mediated by HER2 and its ligand neuregulin 1 (NRG1). Signaling pathways mediated by the ligands LIF and BMP are essential for maintaining the pluripotency of murine ESC, but are insufficient for the self renewal of hESC. The applicants cite several lines of evidence that HER2-mediated signaling may play an important role in regulating the self renewal or differentiation of hESC. Briefly, HER2 and to a lesser extent HER3 are expressed in undifferentiated ES cells. Second, HER2 may be involved in NRG1 and LIF induced activation of STAT3, which may suggest that signaling pathways in mESC and hESC have underlying similarities. Third, there is evidence that GABA is involved in maintaining the undifferentiated stage of hESC via a G-protein coupled receptor (GPCR), and HER2 is known to be a co-receptor for multiple GPCR’s. Lastly, unpublished data from the applicant’s laboratory suggest that type 1 NRG1 can affect the differentiation of neural stem cells, and it is proposed that other NRG1 isoforms could play a role in the self renewal of more primitive stem cells. In Aim 1 the applicant will determine whether different isoforms of NRG1, acting via HER2, are required for self renewal of undifferentiated human ES cells. In Aim 2 the applicant will study the role of this pathway in hESC differentiation into neuronal lineages.
INNOVATION AND SIGNIFICANCE: Insight into the signaling pathways that promote hESC self renewal would aid in the development of strategies for more efficiently propagating these cells in vitro.
STRENGTHS: The experiments proposed in aim 1 are well conceived, although the description of the experiments is lacking in some important details. The applicant proposes three experiments to determine if HER2 signaling is important for hESC self-renewal. First, he/she will determine if exogenous type I and type III NRG1 in defined culture medium is sufficient to support hESC self-renewal as determined by specific protein and mRNA markers for undifferentiated and differentiated states. Synergy with FGF and/or Noggin will also be addressed. Second, he/she will similarly determine the effects of blocking HER2 signaling either by using Herceptin, or by overexpressing a dominant interfering portion of the HER2 extracellular domain. Third, he/she will specifically examine whether HER2 signaling is important for blocking BMP signaling, using exogenous NRGI and NRGIII to activate the HER2 pathway in the presence of exogenous BMP proteins. Signaling downstream of BMP will be measured by phosphorylation of Smad proteins, and by activation of a BMP-dependent luciferase reporter construct.
The specific experiments in aim 2 are also wee conceived. The applicant will ask whether hESC maintained in defined medium with of NRGI and/or NRGIII retains the ability to respond to signals that induce differentiation into neuronal lineages. He/she will look both at differentiation into neuroprecursor cells (which are predominantly cholinergic) and neurospheres (which are predominantly dopaminergic). The applicant will also use these same methods to address whether NRG proteins and HER2 affect differentiation of committed neural progenitors into mature neurons.
The experience of the applicant is strong in molecular biology and murine stem cell biology. The applicant has also focused on a signaling pathway that is likely important in hESC biology and is relatively unstudied in hESC.
WEAKNESSES: The major weakness of the proposal is that the experimental design is poorly presented. Many of the experiments in aim 1 are described in insufficient detail. There are four variables in aim 1 to be tested at different concentrations: Types I and III neuregulin, FGF2, noggin. Dose ranges are given for the neuregulins but not for the other two factors. The reviewer is given no idea how many combinations of concentrations will be tried (or the background as to why these concentrations were chosen) so the Aim reads a bit like a fishing expedition. How many combinations will be used and why?
A list of markers for FACS analysis is given, but the applicant does not state which markers will be used for sorting the cells to be studied in aim 1, part 2, so the plan is unclear. “Cells will be purified by FACS” doesn’t give sufficient information to really see the experimental plan. The same problem with the experimental design of four important factors to be examined (now Types I and II neuregulins and BMP2 or BMP4, both of which have exquisite dose-sensitivity issues) as cited above hold for this part of the proposed work. A minor weakness in aim 1 is that experiments to knock-down expression of HER2 and HER3 in hESC are not proposed.
In aim 2, the applicant will determine the effect of NRG on neuronal differentiation, and also compare the effect of NRG to FGF+Noggin. Aim 2 is to a great degree dependent on the success of aim 1, so the problems in experimental design in aim 1 carry over. A minor problem is that it is not clear whether these assays are quantitative. At no point is it stated how they will measure the efficiency of ES cells differentiation into neural precursor cells or neurospheres.
The proposal would have been significantly more interesting if the role of HER2 as a line between stem cell self-renewal and tumors were looked at in some way after the various manipulations of the hESC.
DISCUSSION: The application is broad in scope, but insufficient in detail. Reviewers liked the concept, regarded the signalling pathway as important but had difficulty figuring out how experiments done.