During human development, autonomic neurons align with and pattern alongside blood vessels. This patterning allows the autonomic nervous system to control the vascular function a phenomenon that is very useful during situations such as "fight or flight" responses where the blood vessels need to respond rapidly and involuntarily to stimuli. Since the alignment of blood vessels with autonomic neurons occurs during embryogenesis, human embryonic stem cells provide a system in which we can observe and understand how neurons and blood vessels differentiate and co-align to form a neurovascular unit.
We have developed a human embryonic stem cell differentiation model where we are able to visualize the early stages of human blood vessel and neuronal development and their co-alignment and patterning in real time over a period of three weeks. Using this model, we have identified a cell-adhesion protein T-Cadherin, present on both the blood vessels and neurons, which may act as the "molecular velcro" in attaching neuronal cells to the blood vessel networks. We have also identified small RNA molecules termed microRNAs that may regulate T-Cadherin protein expression during this process. In this context, we propose that the regulation of T-Cadherin expression by specific microRNAs leads to the differentiation of autonomic neurons and their co-patterning with the network of blood vessels. We will test this hypothesis in three distinct aims:
1. Is T-Cadherin expression necessary and sufficient to drive the formation of the neurovascular unit?
2. Does microRNA regulation of T-Cadherin expression affect neurovascular co-patterning?
3. Can we manipulate T-Cadherin expression to generate viable autonomic neurons from hES cells for possible therapeutic use?
A number of human disorders result from abnormal patterning or development of autonomic neurons. For example, Hirschsprung's disease in infants results from improper nerve development in the gut, leading to chronic bowel obstruction that necessitates immediate surgical intervention. Understanding how autonomic neurons emerge from their precursors and whether aligning with blood vessels are required for their differentiation is critical to develop regenerative therapies. Furthermore, understanding how proteins like T-Cadherin regulate fundamental interactions of different cell types such as blood vessels and neurons offers insights into organ development and tissue engineering.
During human development, specific types of neuronal cells termed 'autonomic neurons' align and pattern along with blood vessels. Autonomic neurons are part of the peripheral nervous system that controls many involuntary functions, including heart rate and blood pressure. Lack of proper autonomic neuronal development or degeneration of existing autonomic neurons can lead to human diseases such as Hirschsprung's disorder, in which improper development of autonomic neurons in the gut results in bowel obstruction. This research proposal aims to understand how blood vessels help the differentiation of autonomic neurons. To this end, our studies will employ a novel human embryonic stem cell differentiation model in which both neurons and blood vessels develop in the context of all three germ layers. The understanding gained from our proposed studies will broadly benefit several categories of patients in California, including those with autonomic nervous system disorders. Furthermore, gaining fundamental insight into neurovascular patterning using this model system will facilitate development of functional blood vessels with proper autonomic innervation for tissue engineering applications that will benefit large numbers of patients in California.
This project will also serve to accelerate innovation of human ES cell and microRNA therapeutics in California, to train more people in California to work on this cutting-edge technology, and to establish the foundation for the design of regenerative therapies for neurovascular disorders that will benefit a significant number of citizens of California.
This application aims to study the cellular and molecular aspects of cross talk between blood vessels and neurons of the autonomous system, which form a neurovascular unit during embryonic development. The applicant proposes three specific aims: 1) to determine if T-Cadherin (T-Cad) expression on endothelial cells and autonomic neurons is necessary and sufficient for their differentiation and co-patterning during human embryonic stem cell (hESC) differentiation; 2) to evaluate the role of a microRNA (miRNA) in regulating T-Cad expression and neurovascular co-patterning; and 3) to determine whether manipulation of T-Cad represents a viable strategy to generate human autonomic neurons for possible therapeutic interventions.
Significance and Innovation:
- The applicant has identified potentially key regulatory factors, specifically the atypical T-Cad and a putative T-Cad regulating miRNA, that may play a significant role in neurovascular co-patterning during early development.
- This proposal fills a niche that has been generally neglected and builds on the substantial accomplishments of the applicant in the field of vascular assembly, maintenance, and growth control.
- The involvement of T-Cad in neurovascular co-patterning and differentiation is based on experimental evidence that is correlative rather than direct, thus lowering reviewer confidence in the underlying rationale.
Feasibility and Experimental Design:
- Proposed experiments are clearly laid out, of sound design, and represent sensible extensions of established methodologies in the applicant╒s laboratory.
- Extensive preliminary data support the technical capabilities of the team.
- There are some concerns surrounding the lack of preliminary data supporting a role for T-Cad in differentiation as opposed to merely promoting co-localization. This question is addressed in Aim 1, but it is not clear that the proposed experiments would provide a definitive answer and a failure of Aim 1 would bring the entire project to a halt.
- The miRNA experiments present some risk. miRNA often have combinatorial effects and, therefore, limiting studies to a single subfamily or a few members might not be sufficient for obtaining meaningful results. The specificity of the miRNA effects on T-cad should be established beforehand.
- Embryoid bodies are not ideal models of embryonic differentiation as they represent random juxtaposition of cell types deprived from morphogenetic movement.
Principal Investigator and Research Team:
- The principal investigator (PI) is a world leader in the field of vascular biology and cancer metastasis biology and has the necessary expertise to carry out the aims of this proposal.
- The research team of is top quality with records of achievement that bode well for success. The environment and resources are superb.
Responsiveness to RFA
- This project utilizes hESC-derived tissues to address the effects of microenvironment on cell fate decisions and is therefore responsive to the RFA. While a single sub-aim will use the zebrafish model to decipher the putative in vivo role of the key factors, that use is justified and appropriate as a correlate, not a substitute, for the in vitro studies on human ESC-derived tissues.