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- MATERIALS AND METHODS
A method of inducing dopamine (DA) neurons from mouse embryonic stem (ES) cells by stromal cell-derived inducing activity (SDIA) was previously reported. When transplanted, SDIA-induced DA neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase (TH) expression. In the present study, to optimize the transplantation efficiency, we treated mouse ES cells with SDIA for various numbers of days (8–14 days). SDIA-treated ES cell colonies were isolated by papain treatment and then grafted into the 6-hydroxydopamine (6-OHDA)-lesioned mouse striatum. The ratio of the number of surviving TH-positive cells to the total number of grafted cells was highest when ES cells were treated with SDIA for 12 days before transplantation. This ratio revealed that grafting cell colonies was more efficient for obtaining TH-positive cells in vivo than grafting cell suspensions. When we grafted a cell suspension of 2 × 105, 2 × 104, or 2 × 103 cells into the 6-OHDA-lesioned mouse striatum, we observed only a few surviving TH-positive cells. In conclusion, inducing DA neurons from mouse ES cells by SDIA for 12 days and grafting cell colonies into mouse striatum was the most effective method for the survival of TH-positive neurons in vivo. © 2002 Wiley-Liss, Inc.
As cell biotechnology develops, cell transplantation therapy is becoming one of the promising treatments for a number of diseases and conditions, including burns, retinitis pigmentosa, and Parkinson's disease (PD). As a source for transplantation, cells derived from patients, human fetuses, and other species such as pigs have been used clinically.
Among neurological diseases, PD is considered to be a good target for cell transplantation. It is a degenerative disorder characterized by a loss of midbrain dopaminergic neurons (DA neurons), with a subsequent reduction in striatal dopamine content. For the replacement of degenerated DA neurons, ventral midbrain cells of human fetuses have been transplanted into the striatum of PD model animals, and this procedure has resulted in the recovery of function by restoration of dopaminergic neurotransmission in the striatum (Brundin et al., 1988b; Olanow et al., 1996). Furthermore, open-label and double-blind, placebo-controlled clinical trials have demonstrated beneficial effects of bilateral intrastriatal nigral grafts, such as increased [18F]DOPA uptake, improvement of Unified Parkinson's Disease Rating Scale (UPDRS) motor score, and decreased therapeutic dosage of L-DOPA (Freed et al., 2001). These clinical outcomes provide a rationale to cell transplantation therapy for PD. However, because of the limited availability of human fetal tissues and ethical problem regarding their use, other sources for donor tissues have been explored. In this context, neural stem cells and embryonic stem (ES) cells have appeared as possible candidates.
Recently, two groups reported methods for generating DA neurons from ES cells in vitro. Lee et al. (2000) reported multistep induction of DA neurons, i.e., the formation of embryoid bodies and expansion of nestin-positive cells, followed by treatment with laminin, fibroblast growth factor (FGF)-2, FGF8, and Shh-N. Kawasaki et al. (2000) reported an induction of DA neurons by stromal cell-derived inducing activity (SDIA) by culturing ES cells on PA6, a bone marrow-derived stromal cell line. This induction of DA neurons by SDIA has proved also to be applicable to primate ES cells (Kawasaki et al., 2002).
For cell transplantation, it is important to optimize the conditions of cell preparation. For this purpose, we induced DA neurons from mouse ES cells by culturing the latter on PA6 cells for different periods and then transplanting cell colonies that formed into the 6-hydroxydopamine (6-OHDA)-treated mouse striatum. In this report, we describe the optimal differentiation period on PA6 and the most suitable condition of the cells for transplantation.
- Top of page
- MATERIALS AND METHODS
In the present study, we examined the optimal conditions for inducing DA neurons from mouse ES cells by SDIA and grafting these cells into the 6-OHDA-lesioned mouse striatum. In those processes, the best period of culturing for ES cells on PA6 monolayers to yield the highest number of TH-positive cells in vivo was 12 days.
Theoretically, TH-positive neurons could have been derived from the grafted cells by two mechanisms. One is that postmitotic TH-positive neurons in the graft survived as they are. The second mechanism is that progenitors of DA neurons in the graft proliferated and differentiated in the brain. In vitro analysis in the present study revealed that the percentage of TH-positive “colonies” reached a plateau by day 12. Nevertheless, the percentage of TH-positive “cells” increased mainly between days 12 and 14. During the process in which the colonies expanded from a few cells, cells committed to the DA neuron lineage may have proliferated in the colony and become postmitotic TH-positive neurons. We infer that commitment to the DA neuron lineage was complete by day 12 but that most of these cells were not mature enough to express TH at that time. In this view, the maturation of the progenitors of DA neurons may have proceeded between days 12 and 14 in each colony.
According to previous reports on the transplantation of ventral mesencephalon from rat embryos in a PD rat model, the optimal donor age for in vivo survival was E11–15 (Björklund et al., 1983; Brundin et al., 1988a). After that period, the survival rate decreased dramatically. This timing is consistent with the time when the first DA neurons of the locus ceruleus and substantia nigra are generated in the rat (Brundin et al., 1988a). This is also the time for DA neuron progenitors in the substantia nigra to undergo their last mitosis (Brundin et al., 1988a; Björklund et al., 1983). In mice, the first DA neurons emerge in the ventral mesencephalon during E10–13 (Di Porzio et al., 1990). It has been shown that the SDIA method mimics the time course of early development of the midbrain (Kawasaki et al., 2000). With all of these facts taken into account, it is reasonable to estimate that ES cell colonies cultured on PA6 for 12 days contain not only mature DA neurons but also a considerable number of DA neuron progenitors ready for their last mitosis. The latter population (DA neuron progenitors) are likely to make good donor cells for transplantation.
The percentage of TH-positive colonies decreased by half after colony isolation. One of the reasons is the damage to the neurites. Interestingly, the decrease in the percentage of MAP2ab-positive or serotonin-positive colonies was much less (about 10%; data not shown). This may indicate that DA neurons are more sensitive to chemical or physical injury than other types of neurons. In focusing on the culture period on PA6 cells, we did not use any cytokines that have neuroprotective or neurotrophic effects. Cytokines such as GDNF (Caldwell et al., 2001; Akerud et al., 2001) or FGF20 (Ohmachi et al., 2000) are reported to have such effects. Their inclusion might help to prevent loss of DA neurons during colony isolation and transplantation.
Another concern in the present study was the cell conditions on transplantation. Grafting cells as colonies resulted in better survival of TH-positive cells in vivo than grafting cells in suspension. One possible reason is a difference in the degree of axotomy of TH-positive neurons between the two conditions. Another possibility is that DA neurons or their progenitors are, as discussed above, damaged by protease treatment. Early studies using human embryonic nigral grafts compared the outcomes of solid grafts and suspension grafts. There was little difference between the two in the survival of TH-positive neurons 4–6 weeks after transplantation into the 6-OHDA-lesioned rat striatum, but the optimal “window” for donor age was different in each case (Freeman et al., 1995; Olanow et al., 1996). Suspension grafts survived best when the donor age was between postconception days 34 and 56, whereas 44 and 65 days were optimal for solid grafts. Therefore, it is possible that more TH-positive neurons would have been observed if we grafted suspensions of cells cultured on PA6 for fewer days, corresponding to earlier embryonic days.
Although we observed no survival of TH-positive neurons from the transplantation of 2,000 suspended cells, Bj¨orklund et al. (2002) reported that transplanting a low density (1,000–2,000 cells) of undifferentiated mouse ES cells into the rat striatum resulted in a proliferation of ES cells and their differentiation into DA neurons. The authors claimed that ES cells in a high concentration often developed into cells derived from all germ layers and that those in a low concentration had decreased cell-to-cell contact and were influenced more by host signals. It seems that undifferentiated ES cells and SDIA-treated ES cells may have different requirements for their response to low-density transplantation.
In this study, 110 TH-positive neurons survived after the grafting of 2.8 × 105 cells. This ratio is lower than that in a previous study by Kawasaki et al. (2000). A possible reason for fewer TH-positive neurons in the present study might be the timing of transplantation after 6-OHDA treatment (3 days vs. 14 days).
If progenitors of DA neurons exist in the graft, as judged from the time course of normal development, a period of 14 days after transplantation seems to be long enough for them to differentiate into TH-positive neurons. However, it is possible that DA neuron progenitors proliferate and remain undifferentiated in the striatum for a longer time. In the present study, we focused on only survival and differentiation of TH-positive cells, but cell migration and neurite extension are also important for an improvement of motor disorders. Although there were no differences of cell migration and neurite extension between cell colonies and cell suspensions 14 days after transplantation in the present study (data not shown), it is possible that cell suspensions have the advantage of better migration throughout the lesioned striatum after a longer period. Furthermore, it would take a longer time for the grafted cells to form a new circuit with host neurons. In consideration of all of this, cell survival, differentiation, tumor formation, and effects on motor function should be examined for longer periods of time to accumulate data for preclinical studies. Now that ES cell lines from monkey (Suemori et al., 2001; Kawasaki et al., 2002) and human (Schuldiner et al., 2001; Zhang et al., 2001; Reubinoff et al., 2001; Xu et al., 2001) sources have been established, similar studies using these primate ES cells and monkeys as a host will be crucial before future clinical studies are performed.