Cell Therapy of Parkinson’s disease with Modified Neural Stem Cells: Explore the Possibilities

Chakraborty A and Diwan A

Published on: 1970-01-01

Abstract

Parkinson’s disease (PD) is characterized by movement disorders, slow speech, and many 21other additional non-motor function disabilities. PD results when a neurotransmitter in the substantia nigra (SN). There is currently no cure for PD available, only management options with DOPA supplementation. However, long-term use of DOPA therapies may result in significant adverse effects, like dyskinesia or motor neuron defects. A number of exciting treatment possibilities may result in near future. One of them is the regenerative cell therapy of PD. However, the main limitation is the availability of sufficient amounts of dopaminergic neuronal cells. Here, we discuss the possible modification method to generate sufficient, functional DOPA-producing cells for regenerative cell therapy of PD.

Keywords

Human neural stem cells; Melanocytes; Cell-Cell interaction; Dopamine; Parkinson’s disease

Introduction

Dopaminergic neurons of the midbrain are the main source of dopamine (DA) in the mammalian central nervous system. Their loss is associated with one of the most prominent human neurological disorders, Parkinson's disease (PD) [1,2]. The disease generally appears at middle age e.g. 50-60, but early onset has also been noticed; it is recognized by slow movement, tremor and with difficulty in normal postures [3,4]. Additionally, depression, anxiety, cognitive decline with dementia, sleep abnormalities are all evident with PD [5]. The real cause of PD is still unknown, although gene abnormalities have been found in 10-15% of PD cases. In other cases toxin exposure an head injuries are found to be involved. In brief, most of the PD cases have no clear cause, hence described still today as an idiopathic disease [6-9].

The number of PD cases in the world is growing noticeably [10]. Furthermore, no such curative treatments are available yet for PD, except palliative treatments like DOPA supplementation as a precursor for DA synthesis [11]. However, longtime use of DOPA as a supplement may cause dyskinesia, motor neuron defects, etc. [12-15].

Therefore, in recent years, instead of DOPA supplementation cell therapy with DOPA-producing cells is being in consideration. Neural stem cells (NSCs), induced pluripotent stem cells (iPSCs), and also other DOPA-producing cells like melanocytes have been studied for using as a cell therapeutic regimen for PD treatment [16-19].

In the recent past there are a number of publications showing the logistics and benefits of doing cell therapy of PD [20-28]. Embryonic stem cells (ESCs) are reported to differentiate into dopaminergic (DA-ergic) neurons, in vitro, using various protocols [29-31]. However the propensity to form teratomas along with ethical issues, limit their clinical uses [32,33]. Midbrain-derived hNSCs may be induced to differentiate into DA neurons, however, the lack of sufficient midbrain tissues is an issue. In addition, midbrain-derived hNSCs lose their proliferative and differentiation capacity in long-term cultures [32].

Human forebrain-derived hNSCs can be expanded in cultures for more than a year without losing their multipotency to differentiate into neurons and glial cells [32]. Thus, those cells can serve as suitable cell sources to provide sufficient number of candidate cells and to differentiate into DA neurons for transplantation uses in PD. More importantly, forebrain-derived hNSCs do not tend to form tumors when used for transplantation [34]. However, forebrain-derived hNSCs appear to have less potential to differentiate into functional DA neurons, which limits their therapeutic application in PD. Gene transfection technology has been reported to produce DA neurons in vitro [35], however there is a safety concern for clinical use of this genetically modified cell type. The therapeutic potential of NSC is broadly accepted by the scientific community but results from the clinical studies performed so far indicate that further studies are needed in order to improve their therapeutic efficacy. However, the in vitro generated iPSC-NSC, although similar to primary neuronal cell lines do not truly recapitulate the phenotype and function of endogenous NSC and retain the risk of tumor formation [36]. In addition, the reprogramming and differentiation processes are lengthy and costly under GMP conditions [36] NewNSC sources and a better understanding of the neurobiology of the cells will surely help in the ambitious quest to 75 repair the brain using cell-based therapies [36].

We have tried previously with hNSCs, primary human neural stem cells derived from the NIH approved H9 (WA09) human (female origin) embryonic stem cells; purchased from ThermoFisher (Rockford, IL, USA) to examine their potential to produce neurons, growth potential and functional differentiation capacity.

However, the problem still remains with their availability in sufficient amount needed for PD cell therapy. This article emphasizes the improvement of the cell quality and quantity, herein hNSCs, by a non-genetic modification method, i.e. cell-cell communication, before transplantation in the PD brain. By cell-cell communication, we mean, either using a cell-cell-co-culturing system and/or cell-cell fusion. However, at present we are focusing mainly on cell-cell co-culture system, not cell fusion. However, we planned to use a 2D cell culture system as our goal to monitor the growth and dopamine secretion by the hNSCs in presence of partner cells hMCs. Similar methods and results have been reported previously using Fibroblasts and HeLa cells [37].

Cell Therapy

Transplantation of a new NSC cell population can restore the lost nigrostriatal DA transmission in PD patients [38]. Various cell lines including genetically modified cells, iPSCs (induced-pluripotent-stem-cells), have been choices for transplantation in PD patients [39,40]. However, there are many issues like possible development of teratomas, ethical issues, cost and time effectiveness of the process makes the choice limited [41-44].

Embryonic stem cells (ESCs) though having the potential to be differentiated into dopamine producing cells in vitro [39,40,44], possess the issues as mentioned above to limit their use in clinics [41,42]. Although midbrain-derived neural stem cells (mNSCs) exhibit the potential to differentiate into Dopaminergic neurons (DA-ergic), their clinical application is restricted due to their low growth potential and multi-potency for differentiation in culture [41]. On the other hand, fetal forebrain neural stem cells (NSCs) can be cultured for a long time without losing their ability to be differentiated into neurons and glial cells [41].

Another neural origin cell-type, melanocytes that are mainly responsible for skin melanin formation from tyrosine, using tyrosinase via DOPA. Therefore, these cells were also proposed for transplantation in PD patients for supplying dopamine in their brain [17-19]. In fact, tyrosinase was found to reverse the PD symptoms in the brain of tyrosine hydroxylase (TH)-null mice [18,45]. Therefore, a therapeutic potential of melanocyte transplantation in the PD brain can be understood. However, melanocytes do not have other DOPA metabolizing enzymes, like Dopamine Transporter (DAT), Mono-amino-oxidase B (MAO-B), and Catecholamine Transferase (COMT), which are needed to catabolize the excess dopamine from the synaptic cleft. The excess dopamine can cause the toxic effects like dyskinesia and motor neuron defect, etc. [12-15,46,47]. Generation of Tyrosine Hydroxylase-positive neurons from human Embryonic Stem Cells were shown after co-culture with cellular substrates and exposure to Glial cell line-derived neurotrophic factor (GDNF) [48,49]. These modified cells also express DAT (Dopamine Transporter), COMT, and MAO-B that together can maintain the physiological level of dopamine in the synaptic cleft [50,51]. Therefore, hNSCs can be considered as a better choice over the other cell types for cell transplantation therapy of PD patients [52]. However, slow growth of hNSCs and senescence after a few passages hinders the ability to procure enough cells needed for transplantation. We are trying to test whether the hNSCs can be modified by co-culturing with melanocytes for their increased growth potential, survival length along and their DA-ergic ability. In brief, for cell replacement therapy of PD, our goal is to select or modify any existing potential cell type for their moderate growth potential, differentiation capacity, axon production and DA-ergic ability. Lastly, the cell should metabolize any excess dopamine from the synaptic cleft to prevent the possibilities of hallucinations, motor neuron defects and other side effects.

Cell-Cell Interaction, a Method of Cell Modification

Cell-cell interaction has been reported to modify each cell in the interaction [53]. This process of communications in between two cells is well evident in somatic cell biology in vivo and in vitro, and in cancer cell biology, as well [54,55]. Cancer cells can interact with their surrounding macrophages, and that communication results in either tumorsuppression or progression depending on the type Macrophages interacting with the cancer cells [56-61]. Cancer-associated fibroblasts can also promote growth and progression of the cancer cells [62]. Furthermore, cancer cells on interaction with endothelial cells can elongate blood vessels [63]. Overall, cell-cell communications mutually can alter the phenotypes and genotypes of each other [64].

Intercellular Communication as a Method for Neural Cell Modification

Like immune cell synapses, the classical neuronal synapses are involved in cell-cell adhesion and membrane association. Studies have shown that the release of serotonin (5-HT) in mice as well as in humans results from the interaction of dendritic cells (DC) and the naive T-cells [65,66] Together, many biological functions such as, embryonic development, neurotransmission, wound healing, inflammation, etc. are all shown to result from cell-cell interaction [60,67].

Cell–Cell Interactions for Neuron Formation

Neurons and axons interact with each other in normal neuro-circuitry pathways [68]. Under ischemic conditions, microglial phagocytosis of apoptotic neurons were induced by remodeling fractalkine receptor (CX3CL1/CX3CR1) mediated signaling [69]. NSPCs (neuronal stem progenitor cells) after activation migrate to the ischemic lesion and proliferate there [70]. Many other incidences of similar of cell–cell communications under stress were also reported. In glial cells, metabolic deficits in axonal energy are related to neurodegenerative diseases, like PD and AD [71]. Oligodendrocytes release lactate to neurons through MCT1 protein (monocarboxylate transporter1), and it’s dysfunction causes neuronal/axonal damage [72]. In fact, deficiency of MCT1 leads to a disruption of the trophic support to neurons (axons) found in an ALS (amyotrophic lateral sclerosis) mouse model as well as in human subjects [73].

Cell-Cell Interaction Can Effect Dopamine Production

Dopamine is the main neurotransmitter released by NSCs in brain, responsible for movement, endocrine regulation and cardiovascular function, as well as in maintaining the cognitive functions [74,75]. Further, dopamine is the main precursor of adrenaline and noradrenaline hormone releases in the nerve periphery [76]. Leukocytes, which are abundant in surrounding NSCs, release dopamine which may act as an autocrine and paracrine immune modulator [77]. The synthesis and storage of dopamine in monocyte-derived 168 dendritic cells (Mo-DCs) can be enhanced by cAMP producing cells [78]. Likewise, DA-ergic cells can increase cAMP levels in CD41+ T cells [CD4+T cells recognize peptides presented on MHC class II molecules, which are found on antigen presenting cells (APCs)], indicating a possible cross-talk are there for mutual benefit.

Partner Cell Selection for Interaction with hNSCs

The followings are the cells that can be considered for interaction with hNSCs, but the question is which one should be the best choice for the purpose. Table 1 describes the different cells and their merits and demerits in using for modifying the NSCs.

Table 1: Cells with their Merits and Demerits in using for Modifying NSCs.

Human Embryonic Stem Cells (hESCs)	Neural Progenitors Cells (NPCs)	Bone Marrow Derived Adult Progenitor Cells	Melanocytes
ES can modify the growth potential of other cells [79]	NPCs are multipotent [84]	Bone marrow derived adult progenitor cells can trans-differentiate and generate neurons in vivo [87-90]	Melanocytes are originated from neural-crest, and committed to form skin melanin [90,91]
hES cells can modify the growth potential of hNSCs by cell-cell interaction [80]	NPCs can migrate, differentiate into neurons and can make synaptic contacts with the local neurons [84,85]	Stem cells may not have originally the neurons but can extends a new neuron on fusion with the host neurons [88,89]	These cells express melanocortin receptor (MC1) and releases the second messenger, cAMP [91]
The possibilities of forming teratomas and ethical issues limits the use of hES cells [81-83]	Use of this cell for clinical purpose, however, is still in dispute [85-87]		cAMP and/or its inducers can increase dopa and dopamine production, in vivo and in vitro [92,93]
			Dopamine or its precursor dopa can increase the cAMP level through D1-receptor-mediated signaling [92,93]
			Therefore, a cross-talk between the neural cells and melanocytes can be expected.

Our Approach

From the above information, Melanocytes can be considered possibly as a better partner cell type over others to modify the growth potential of NSCs. We are interested to study, in particular, whether melanocyte can influence neural cells for their proliferation, increased survival length, along with other inherent properties like, axon production, dopamine and growth factors BDNF/GDNF secretion.

We have studied cell–cell interaction by co-culturing human normal melanocytes (hNMCs) with human Neural Stem Cells (hNSCs) in vitro [94,95]. hNMCs primary normal human adult melanocytes, lightly pigmented, from male origin were purchased from ATCC, Manassas, VA, Cat# PCS-200-012). These cells produce and secrete melanin. The source and type of human Neural Stem Cells (hNSCs) are already mentioned above in Section 1 [94,95].

This co-culture method is quite similar to co-culturing cancer cells andendothelial cells, fibroblasts and immune cells, reported previously [37]. In brief, we used the 2D culture system according to the reference citations, which differs from 3D culturing system in many ways but still possesses several advantages [For comparison, see Ref: 96]. Although the 3D cultring system mimics mostly the in vivo condition, it cannot exchange nutrients and waste freely like 2D culture. Furthermore, 2D cell cultures are easier for environmental control, observation, imaging, measurement and eventual manipulation, if needed.

Documentation of cell growth, cell morphology and axon production at different time points were done. Expression levels of NSCs-specific proteins like, Nestin, Tubulin III (Tuj1) were studied using immuno-fluorescent microscopy. ELISAs were done for measuring the secretion of Dopamine, BDNF and GDNF by NSCs. In brief, co-culturing of hNSCs with hNMCs in vitro showed an increased growth potential of hNSC’s, survival length, and increased capability in producing dopamine, BDNF/GDNF as reported earlier [94,95].

Discussion

The human body is composed of different types of cells with specialized functions, and they do communicate with other cells from different organs [97, 98]. This communication occurs either through direct physical contact, receptor-ligand interaction, cellular junction, or via secreted materials like proteins, cytokines, metabolites, growth factors, etc. from the neighboring cells [99,100]. Interactions via extracellular vesicles are also a possible way of communication [101]. Furthermore, physical properties of the surrounding extracellular matrix, hypoxia and energy level modulate cell-cell communication [102]. In humans, LO–LO (lipoxygenases) interactions positively modulate the biosynthesis of Lipoxin (LX). LXs are a local mediators produced from arachidonic acid responsible for distinct and potent biological roles in inflammation, infection, or injury [69,103].

Together, it is conceivable that cell-cell interaction can contribute for biological functions of the organism, such as, embryonic development, wound healing, neurotransmission, inflammation, etc. In the scenario of Parkinson’s disease (PD) where the loss of DA-ergic neural cells in substantia nigra (SN) region of the brain was evident, the replenishment of cellular loss with a modified neural cells, is expected to be a valid curative approach. Furthermore, we can predict from our co-culture experiment that melanocyte and NSCs can lead to an autocrine-paracrine communication for better growth of NSCs and their secretion of dopamine. Dopamine, in turn, may induce cAMP formation from melanocytes and that 2^nd messenger can further induce the neighboring melanocytes to produce DOPA, a precursor of dopamine produced by neural cells.

Acknowledgment

We acknowledge all of our staff members, scientists from AllExcel, Inc. Especially Ms. Beth Pond to support during the writing of this review by providing materials, and editing.

Authors Contribution

All the authors contributed equally.

Conflict of Interest

This research is supported by internal grant from AllExcel, Inc. The authors report no conflicts of interest.

International Journal of Stem Cells and Regenerative Medicine