In-Vitro Differentiation of Human Stem Cells into Dopaminergic Neurons

Abstract

The inability of the neurons to regenerate has encouraged the scientists to search for a method to replace damaged or dead nerve cells. Parkinson disease (PD) is the most common neurodegenerative disorder, affecting 2% of individuals over age 65 and 4-5% over 85 years, and it is caused by the loss of dopaminergic (DA) neurons. There is no effective treatment for PD and the disease progression cannot be counteracted, as well as over time the side effects of medications appear. Therefore, nerve cell replacement is an important therapeutic option for PD.

The aim of this study was to explore the in-vitro ability of human MSCs derived from peripheral blood, after their mobilization from bone marrow, to differentiate into DA neurons.

Peripheral blood mononuclear cells (MNCs) were obtained from 10 healthy donors undergoing stem cell mobilization for stem cell transplantation after confirmed verbal consent. The age of the donors ranged from 25 to 35 years. They were 8 males and 2 females. The donors were subcutaneously injected with Granulocyte-colony stimulating factor (G-CSF) in a single daily dose of 10 µ g/kg for 5 days. Then collection of MNCs by apheresis was done on the fifth day one hour after injection of the last dose.

Total leucocytic counts were done before and after mobilization. Culture and separation of MSCs were done. Cell viability test was done after culture, and immunophenotyping of CD44 cells was done before and after culture. Differentiation of MSCs into neural lineage was done using nerve growth factor, while differentiation into DA neurons was done after using ascorbic acid. Immunostaining was done using anti neurofilament (NF) and anti tyrosine hydroxylase (TH) antibodies.

Colonies of MSCs were detected by the inverted microscope 3 days after culture and they increased markedly after 7 days. Cultured specimens immunostained with anti NF and anti TH antibodies showed scattered immunopositive cells (brown deposits). The percentage of cells stained for NF was 15.38±3.84, while the percentage of cells stained for TH was 5.94±0.65.