Summarized by: Sandra Hong and Michael Poynton, Fall 2007

 

LAY SUMMARY

Embryonic stem cells (ESCs) have been proposed for the treatment of Parkinson’s Disease (PD) as well as experimental models. Despite their potential, there are scientific problems with ESCs. They are difficult to control mainly to their ease to commit to cells of three germ layers. This uncontrolled growth often leads to unwanted tumor or teratoma formation.  The researchers of this article reasoned that using a mixed population of ESCs would more likely result in teratoma formation than a purified population.  Although fewer dopamine neurons are acquired from a purified population, the researchers argued that teratomas overshadow any behavioral improvement in the PD model.  For this reason, they attempted to eradicate the possibility of teratoma formation by removing the undesired proliferating cells and select for an ideal cell population to derive dopamine neurons. The selected neurons are intended for transplantation into animal model of PD. 
                            
The selection was done with rat tyrosine hydroxylase (TH) promoter, which drives the enhanced green fluorescent protein (eGFP) gene. The objective is to select the cells forming dopamine, which would be identified by green fluorescence. Two different TH promoters were tested based on size. The goal was to determine which promoter would be more efficient in the identification of TH positive cells. It was found that the 9 kilobase (kb) promoter produced more eGFP+/TH+ neurons than the 2-kb promoter. Hence, the 9-kb clone was chosen for subsequent experiments. 

Transplantations of promoter-expressing mES cells were performed on 15 C57/Bl6 mice and 18 Sprague-Dawley rats.  While the mice were naïve or untouched, the rats were administered 6-hydroxydopamine, a neurotoxin that induces the symptoms of PD.  Another group of rats were given 6-hydroxydopamine lesions and medium (vehicle) alone as opposed to a graft of 9 kb TH-eGFP mES cells.  To measure restoration of function in the PD model, the researchers used amphetamine to observe rotational behavior. 

Although the intentions of the researchers were good and promising, the outcome of their experiments was ambiguous. In all grafts, eGFP+ cells and TH neurons were present. Furthermore, most TH neurons also expressed eGFP. Unfortunately, the positive result was dampened by the presence of a mixed population of cells.  The effort made to achieve a purified population still resulted in unwanted phenotypes.  The cells were composed of those that expressed only TH or only eGFP.  Some of the cells in the resulting population lacked neuronal morphology. This negated thee expectation to select only germ layer cells consistent with dopamine positivity. However, the authors found cells of all three germ layers, indicating that the GFP showed non-dopaminergic cells. Teratomas were disappointingly not avoided.  Several rats exhibited behavioral problems that indicated teratoma formation.  From the remaining rats that did not form tumors, it was found that there was a minor improvement in rotational behavior (less rotating), but it was not significant enough of an improvement to associate it with the number of TH+ neurons. To address the perplexing cell population, the researchers offered a possible explanation by stating that TH gene activation may be transient.  TH expression may be turned on during certain time points of development and turned off in other stages. This explanation illustrates how difficult and almost impossible it is to use a single marker to select for a population of embryonic stem cells. The article proves that stem cell biology is very dynamic, and the selection process to avoid teratoma formation with ESCs needs further improvments.         

 

SCIENTIFIC SUMMARY

The researchers involved in this article were seeking ways to purify dopamine neurons from embryonic stem cells for purposes of transplantation into naïve murine and Sprague-Dawley 6-hydroxydopamine-lesioned rat Parkinson’s disease models.  It is already widely known and accepted that during embryonic stem cell manipulation and transplantation, they have the potential to proliferate uncontrollably and subsequently produce cells and tissues from all three germ layers in a disorganized manner.  This often results in the development of a teratoma in animal models, and there is subsequently no therapeutic benefit achieved.  With this caveat of embryonic stem cell transplantation in mind, the researchers hypothesized that “purification of dopamine neurons derived from embryonic stem cells by fluorescence-activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation.1”  In this experiment, mouse embryonic stem (mES) cell-derived dopamine neurons were used and they essentially tried to pre-determine which mES cells would have the greatest potential of success prior to transplantation, and then monitor their engraftment and integration into the striatum of the brain.

Among the different methodologies and techniques that could have been implemented, the researchers decided to use a tyrosine hydroxylase (TH) promoter in order to drive enhanced green fluorescent protein expression (eGFP) in the mouse embryonic stem cells.1  The researchers were aware that transgenic expression of enhanced green fluorescent protein from the TH promoter could be used to purify primary dopamine neurons from embryos after sorting by FACS for transplantation.1  Thus, continuing with an already proven method, the authors applied the technique in the current studies.

The authors tested various size TH promoters to determine which one would produce the greatest number of eGFP positive and TH positive neurons.  In layman’s terms, you need a promoter fragment before gene expression can occur and produce gene products such as dopamine.  After extensive generation of clones and observing their in vitro differentiation prior to any transplantation experiments, it was found that a 2.5 kb promoter fragment only generated 10 out of 108 clones that were eGFP positive neurons, and only 2 of these 10 clones exhibited TH positive neurons.1  On the other hand, a 9 kb promoter fragment generated 85 out of 108 clones that were eGFP positive neurons and greater than 10 of these 85 also had TH positive neurons.1  In summary, the 9-kb eGFP/TH clone which showed higher co-expression of  TH and eGFP was selected and used for the remainder of the experiments including the transplantation studies.  In addition to the promoter size fragment, the researchers were required to identify the most efficient in vitro-based protocol. Between the MS5 and PA6 protocols, they quickly determined the MS5 protocol generated the greatest percentage of eGFP-positive cells and eGFP+/TH+ cells, which was critical to the studies.

In order to get these pre-purified or pre-selected embryonic stem cell-derived enhanced green fluorescent protein-positive dopamine neurons into the mice and rats, the authors applied a previously reported transplantation procedures (2).  The animals were injected with 1.0 µl of ES cell suspension (1,000-2,000 ES cells per µl) or vehicle into two sites of the right straitum. The injection used 22-gauge needles attached to 10-µl Hamilton syringes. A 2-min waiting period allowed the ES cells to settle before the needle was removed. Overall, after short-term transplantation, eGFP positive cells with large numbers of TH neurons were observed in all grafts. Most of the TH positive cells also co-expressed eGFP, although some cells were found to be either TH positive or eGFP positive (1). Many of the eGFP positive neurons displayed characteristics of midbrain dopamine neurons, based on co-expression of AADC or aromatic L-amino acid decarboxylase. The studies attempted to provide an environment that would enrich for midbrain dopamine neurons while minimizing the generation of norepinephric neurons or non-neuronal TH cells.

The researchers were not 100% conclusive that they obtained dopaminergic neurons. However, the authors observed some improvements with respect to Parkinson’s symptoms, although their results were somewhat contradictory. The 6-hydroxydopamine-lesioned rats would rotate around in their cages in a direction that is towards the side of their body in which the lesion exists.  Observing their rotational behavior before and after transplantation, they thought they would be able to gain insight into their biological improvements. They noted that amphetamines administered to the rats, could induce improvements in rotational behavior without there being any morphological integration of transplanted cells.  This lack of correlation between what the researchers’ observation and the actual number of TH+ cells in the brain bring to question the visual data.

A significant observation was the presence of higher numbers of stage specific embryonic antigen positive (SSEA-1+) cells in the genetically engineered and expanded clones versus in the original mouse embryonic stem cell (mES) cell line after differentiation, indicating malignant transformation (1).  The authors could not determine the exact conditions that led to SSEA+ cells. There were less of these cells present in the original mES cell line before any manipulation ever took place than there were in the in vitro differentiated cells. They checked for chromosome abnormalities, trying to gain evidence as to why these cell events were occurring but could not provide anything other than mere speculation. 

The authors stated that some of the integrated clones were dopaminergic in nature because these clones responded to dopamine differentiating signals, while others were unresponsive. One of the original goals of preventing teratoma formation, was not completely achieved as some of the animals had to be sacrificed early. The door has been left open to continue research, and the researchers admitted that a single marker for selection of a specific cell population in embryonic stem cell cultures might not be efficient. To somehow eliminate some of the variables that came into focus during the discussion of the article and to be able to reduce the experimental parameters so that a correlation could be possibly made, might provide a more stepwise but thorough answer.

 

References