Molecular basis of human ES cell neurovascular differentiation and co-patterning

The development of a blood vessel network that can respond to autonomic neuronal inputs is a critical achievement during organogenesis. This system confers a functional benefit by delegating the involuntary control of vascular function to the autonomic nervous system, a phenomenon that is best illustrated by the “fight or flight response”. During the past year, we have focused on understanding the requirement of a particular cell adhesion molecule for proper co-patterning between blood vessels and neurons. Using several in vitro models, we were able to observe the timecourse for the development of vascular tubes comprised of multiple cell types. We discovered that co-patterning between vascular endothelial cells and autonomic neurons requires the presence of third cell type, vascular smooth muscle cells. Furthermore, we have identified a critical cell adhesion molecule that facilitates the interaction between these three cell types. Our studies focused on how autonomic neurons emerge from their precursors and our finding that aligning with blood vessels helps their differentiation is highly relevant for the development of new regenerative therapies for diseases of the autonomic nervous system, and ultimately for the engineering of organs with functional vascular networks.

The development of a blood vessel network that can respond to autonomic neuronal inputs is a critical proper organ formation and function. During the past year, we have focused on understanding the requirement of a particular cell adhesion molecule for proper co-patterning between blood vessels and neurons. In the past review cycle, we found that two different vascular cell types (endothelial cells and vascular smooth muscle cells) were required to drive stem cell differentiation towards a neuronal fate. In the past year, we showed that a direct cell-cell interaction between vascular smooth muscle and stem cells is required for their differentiation toward an autonomic fate. We found that this is due to a homotypic interaction between critical cell adhesion molecules expressed on the surface of both cell types. Understanding how autonomic neurons emerge from their precursors by aligning with blood vessels to drive their differentiation is highly relevant for the development of new regenerative therapies for diseases of the autonomic nervous system, and ultimately for the engineering of organs with functional vascular networks.

Our overall goal was to understand how blood vessels and neurons come to be co-aligned in the human body, a process which we will refer to as “neurovascular co-patterning”. Generally, stem cell research focuses on the derivation and differentiation of single lineages in isolation from other cell types. However, the true vertebrate body plan is not constructed in that way. Indeed, most biological processes – starting with the earliest stages of organogenesis in the embryo and extending to organ regeneration in the adult -- demand exquisitely coordinated co-patterning of multiple lineages (often derived from different embryonic germ layers) for function to be normal. Neurovascular co-patterning (i.e., alignment of nerves and blood vessels) is a prototype for such a developmental program. By examining neural and vascular co-development in a human embryonic stem cell model, we offer a mechanism by which such co-patterning might emerge and in which primitive blood vessels then play an active role in early neuronal specification and differentiation. Neural crest cells are a type of stem cell that can differentiate (i.e., to become more specialized) into one of many neural or non-neural cell types. We provide evidence that neural crest cells respond to distinct cues from the cell types comprising blood vessels: endothelial cells and vascular smooth muscle cells. Specifically, these signals prompt the neural crest cells to specialize into a particular kind of neuronal cell called an autonomic neuron. These are the cell types that control basic body functions below the level of consciousness, such as the control of blood flow. We discovered that the process of neurovascular co-patterning requires two independent events. First, the neural crest cells must respond to a factor produced and released by the blood vessels, called “nitric oxide”. This signal provides a molecular cue facilitating neural crest cell differentiation towards an autonomic fate, as opposed to all of the multiple non-neural fates to which it might also have been directed. Secondly, this process is then further refined by direct contact between neural crest cells and the cells lining the outside of each blood vessel (“vascular smooth muscle cells”). An interaction with a cell adhesion molecule present on both cell types (“T-cadherin”) solidifies the fate of the neural crest cells. Understanding how this process occurs and which elements are required will allow us to develop a new method to generate autonomic neurons in culture, a still elusive goal of regenerative medicine. Further development of this strategy will serve a significant unmet medical need and facilitate development of reparative therapies for dysautonomias such as Hirschsprung’s disease.