A long and winding road: Differentiating human embryonic stem cells into dopaminergic neurons

Abstract

Parkinson’s disease is a progressive neurodegenerative disorder. Since the motor manifestations of the disease is associated with the loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta, cell replacement therapy is a promising approach for treatment. Although several different cell types have been used in clinical trials, with the recent findings of somatic cell nuclear transfer in humans, human embryonic stem cells (hESCs) offer a promising “personalized” source of cells in this quest. In the present study, we established a simple protocol for large-scale generation of dopaminergic neurons from hESCs using PA6 cells as feeders. We showed that the intermediate cell type, DAergic neural progenitors (NP), generated during the differentiation could be expanded for long time and cryopreserved without loosing their full capacity to differentiate into DAergic neurons. We further revealed that within the heterogenous PA6 population, adipocytes robustly express all the factors previously identified to be associated with SDIA. Coculturing with PA6-derived adipocytes reliably promoted the dopaminergic differentiation of the hESCs. Moreover, we identified three candidate molecules that might be involved in the SDIA of PA6 and PA6-derived adipocytes. We also demonstrated that S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) efficiently induced apoptosis in proliferating neural progenitors through the TRAIL pathway, and promoted the DAergic differentiation of the hESCs. Additionally, we also identified twelve candidate surface markers that can be used in MACS for purifying DAergic neural progenitors within the heterogeneous population.

Abstract (Swedish)

Popular Abstract in English

Parkinson’s disease is the second common neurological disease whose major clinical symptom is progressive impairment in the ability to control the body movements. The symptoms are mainly caused by the gradual loss of dopamine producing neurons in a very specific part of the brain, termed substantia nigra.



Although there exist some drug and surgical therapies, those mostly introduce some serious side effects and their alleviation on the disease is mostly temporary. Therefore, replacing the dead neurons with the healthy ones by transplanting new dopamine producing cells is a promising approach for the treatment of the Parkinson’s disease. Indeed, previous clinical trials using dopamine-neuron-rich tissues obtained from the aborted fetuses proved that this approach might work; however, using fetal tissue is controversial and some other studies had highly variable results. Several other cell types from different sources have also been tested which gave similarly mixed results.



One of the most promising cell sources for replacing those neurons is human embryonic stem cells (hESCs). Embryonic stem cells can be cultured for long periods multiplying their number indefinitely and they can also be converted into all cell types of the body including the dopamine producing neurons. However, currently their conversion into dopamine neurons is not efficient. Regardless of which method is used, in the end there is always a mixed cell population composed of some resistant hESCs remaining that could not been converted, some dopamine producing neurons and some other cell types in between. There are several approaches to overcome this problem. Considering that we currently do not fully know the exact mechanism how this conversion of hESCs into dopamine producing neurons, understanding this mechanism fully will help scientists to establish better protocols for efficiently converting hESCs into dopamine producing neurons.



The most commonly applied method for this conversion is culturing hESCs with the support of some other cell types which mostly originated from bone marrow. Those cells secrete some molecules and growth factors that induce hESCs to become dopamine neurons. However, those “feeder” cells are also a mixed population of cells which makes it hard to reveal secreted molecules as there are additional unrelated molecules in the mixture secreted from these other cells. In this study, we demonstrated that within this mixed feeder cell population, adipocytes are the real cell types that efficiently produce the previously identified factors required for the dopamine neuron conversion of hESCs. Further studies concentrating on this very specific cell type and revealing the molecules it secrete will accelerate the quest for identifying and fully understanding the exact mechanism for the conversion of hESCs into dopamine neurons. We additionally revealed three new molecules secreted in adipocyte cultures that might be involved in this dopamine neuron conversion of hESCs since they were previously shown to have beneficial effects on dopamine neurons.



Another promising approach for getting highly pure population of dopamine neruons converted from hESCs is selecting those cells individually among the mixed population. Since it is impossible to do that by hand, there exist several methods for selecting specific cells in the mixed population. Among those methods, marking the desired cell type using magnetic beads and and then catching those marked cells with a magnet is considered the most gentle method for dopamine neurons. In this study we tested three specific markers for selecting those cells; however, in our hands we observed a major cell death after the cell sorting process using those markers suggesting they might not be suitable for selecting the cells using magnetic beads. Thus, we identified 12 novel candidate markers for further studies.



One of the biggest problems in the dopaminergic conversion is the remaining embryonic stem cells at their original state as well as some other highly dividing cells in the end of the process. Indeed, such cells was shown to form tumor or teratomas when transplanted into the animals. Embryonic stem cells share many similarities with cancer cells. Therefore, we tested a chemical compound, named DZNep, that was previously shown to selectively kill the cancer cells but not normal cells. Our results revealed that, DZNep also effectively killed the hESCs and highly proliferating cells, but not other cells. Moreover, DZNep treatment increased the proportion of the dopaminergic neurons generated from hESCs.



It is also important to generate dopamine neurons in large numbers to be sufficient for transplantation, drug screening and toxicology assays. Unfortunately, most of the protocols established so far do not fulfill this requirement. In this study, we also established a novel protocol for large-scale generation dopamine neurons from hESCs. We could generate an intermediate-stage cell type, called neural progenitors that could be expanded for months multiplying its number at least 5x106 times, could be stored in ultra-low temperatures for years and could further be converted into dopamine producing neurons within few days when needed.

• author: Guloglu, Oktar
• supervisor: Jia-Yi Li
• opponent: Prof Caldwell, Maeve, University of Bristol
• organization: Department of Experimental Medical Science; MultiPark: Multidisciplinary research focused on Parkinson´s disease
• publishing date: 2015
• type: Thesis
• publication status: published
• subject: Neurosciences; Cell and Molecular Biology
• keywords: adipocyte, PA6, DZNEP, dopaminergic differentiation, embryonic stem cells
• in: Lund University Faculty of Medicine Doctoral Dissertation Series
• volume: 2015:80
• pages: 260 pages
• defense location: Segerfalksalen
• defense date: 2015-08-26 09:15:00
• ISSN: 1652-8220
• ISBN: 978-91-7619-159-0
• language: English
• LU publication?: yes
• id: ea7ead1e-282a-4360-9767-b49e00df29a0 (old id 7758127)
• date added to LUP: 2016-04-01 14:37:49
• date last changed: 2019-09-09 14:10:30