Differentiation of Peripheral Sympathetic Neurons

Abstract

This thesis is a literature review of the methods that have been used to differentiate sympathetic neurons in vitro from human origin stem cells. Information from the previous studies was collected and different methods used to differentiate sympathetic neurons were compared and discussed. Focus was also on the possibilities to use sympathetic neurons in co-cultures with cardiomyocytes to model human body functions.

More effective sympathetic neuron differentiation methods have been developed over the years. The aim of the first studies performed with human stem cells was to differentiate neural crest stage cells. At first, embryonic stem cells were utilized, and the methods were based on co-cultures with stromal cell lines and use of few specific factors to guide the differentiation. The methods have been based on mimicking the signaling pathways of neuronal development during embryonic development. Later methods counted more on so called small molecule methods and induced pluripotent stem cells have been preferred over embryonic stem cells. Generation of sympathetic neurons from stem cells takes on average one month of cell culture. The yields of sympathetic neurons and their characterization has been improved with modern methods.

The most important factors in the sympathetic neuron differentiation were bone morphogenic proteins and molecules or drugs that can regulate for example Wnt- and SHH-signaling pathways. In the most recent and the most effective differentiation studies, three small molecule inhibitors and different variations of them were used. Precise growth conditions are needed to achieve and maintain cultures of differentiated peripheral neurons. Sometimes cell sorting by specific markers was used during differentiation steps to enhance the differentiation efficiency, but to simplify the methods, it is not preferred. The markers that were most commonly used to characterize sympathetic neurons are peripherin, tyrosine hydroxylase, dopamine beta hydroxylase and PHOX2B gene expression.

In addition, this work presented how differentiated sympathetic neurons are already used in co-cultures with cardiomyocytes. Thus, it is possible to confirm neuron functionality and to model human body interactions of these cells. Results of this work clarified well the literature concerning sympathetic neuron differentiation and the most effective methods for the differentiation were recognized as an outcome.