Numerous studies have demonstrated the use of ESC-derived DA neurons for grafting in animal models of PD (17
); however, it has remained unclear which stage of differentiation and what particular population of cells is most effective at inducing functional benefits upon transplantation in vivo. Fetal tissue studies have focused on the grafting of ventral mesencephalic tissue isolated at a stage in which the majority of developing DA neurons are postmitotic, which is E12–E13 in mice (18
) and E14–E15 (19
) in rats. In human fetal grafting studies, increased survival of TH+
neurons has been reported for tissue derived from embryos at 5 to 8 weeks after conception when using suspension grafts and up to 9 weeks after conception for solid grafts (20
). In all of those cases, however, the grafted population was composed of a heterogeneous pool of cells at various stages of differentiation and typically contained less than 20% DA neurons (21
). One recent study used purified DA neurons derived from a Pitx3::GFP
plasmid reporter line established by gene targeting (4
). The authors reported a requirement for coculture with primary astrocytes to achieve robust in vitro survival of FACS-purified DA neurons and observed less robust in vivo survival rates. Nevertheless, the study did demonstrate functional engraftment of purified DA neurons in at least a subset of animals (4
In this study, we observed excellent in vitro and in vivo survival for DA neurons derived from both the Nurr1::GFP
lines without astrocyte coculture, thus providing clear evidence that transplantation of purified early-stage DA neurons is a promising option for PD grafting. The requirement for double sorting to eliminate tumorigenic cells was associated with a lower survival rate of TH+
cells. While animals with double-sorted grafts received half the number of cells compared with those with single-sorted grafts (due to our limited scalability), TH+
cell survival in double-sorted grafts was 10 times lower. This lower DA neuron survival rate could be due to factors secreted by contaminating cells in single-sorted grafts or increased mechanical stress involved in the double-sorting process or point to a critical threshold of total cells needed for graft survival and integration. Despite the decreased survival rate, the number of DA neurons delivered via double sorting was sufficient to provide therapeutic benefits in our behavioral assays. It has been shown that as few as 400 TH+
neurons can provide up to 50% reduction in behavioral deficits of hemiparkinsonian rats 6 weeks after surgery (22
cell numbers in our study clearly surpassed this threshold in both single- and double-sorted populations, and we commonly observed contralateral rotations in our single-sorted grafts following amphetamine stimulation, a finding in often interpreted as overcompensation. Among the 3 stages of DA neuron development tested here, in vivo TH+
cell yields were highest in reporter+
cells from the Nurr1::GFP
line, without evidence of reduced specificity in DA neuronal fate. Therefore, transplantation of purified early postmitotic DA neurons at the Nurr1+
stage may be a particularly promising approach for eventual clinical translation. However, it should be noted that recovery of behavioral deficits was not statistically significant in the cylinder or apomorphine tests. This could be due to various factors including limited fiber outgrowth of the DA neurons; low levels of DA release; or technical limitations of our study, such as measuring performance in only a single test both before and after grafting, low sample size, and limitations of the mouse as opposed to the more commonly used rat 6-OHDA model. However, in terms of recovery of amphetamine-induced motor deficits and overall DA neuron cell survival of single- and double-sorted grafts, we found that the Nurr1
stage of differentiation is ideal for grafting ESC-derived DA neurons.
Future studies will be needed to address the effect of glial cells, or lack thereof, at the time of grafting. Our data indicate that graft-derived DA neurons are surrounded by glia from the host. But currently it is unclear whether the host glia help or hinder DA neuron survival and function. In double-sorted cells, there was a trend toward a more robust behavioral recovery in Hes5::GFP+ grafts compared with that in Pitx3::YFP+ grafts, despite similar or higher numbers of TH+ cells in the Pitx3::YFP group. One potential explanation could be that the presence of graft-derived astrocytes from the Hes5::GFP line promotes DA neuron function. Future studies will be needed to assess the survival and function of FACS-purified DA neurons in the presence or absence of astroglia.
The present findings underscore the need to graft populations highly enriched in neuronal fates in order to prevent tumor formation. When grafting cells at high concentrations in an immunocompromised host brain, we found that a percentage of less than 1% contamination (175–1,600 SSEA1+
cells) at the time of grafting represents an unacceptable tumor risk. In addition to the double-sorting technique presented here, we could also largely prevent tumor formation by gating cells that were negative for SSEA1 expression during the initial reporter+
cell sort, similar to previous work (23
). We found the SSEA1-647 antibody to be particularly effective in reducing SSEA1 contaminants after sorting (Supplemental Figure 3). Attempts at reducing potentially tumorigenic cells prior to transplantation via short-term replating could decrease, but not fully eliminate, SSEA1+
cells (Supplemental Figure 3, C and F), which is in agreement with the more rare overgrowth behavior observed in previous work (2
). Tumor formation from SSEA1+
-grafted cells occurred within 2 weeks following transplantation in our study, which is in contrast to the much slower time course observed in hESC-based neural grafts that more commonly show neural overgrowth rather than teratoma formation (1
). The differences in teratoma risk between mESCs and hESCs may be related to their respective pluripotent states, with hESCs being developmentally aligned to mouse epiblast stem cells (24
), which are more primed for differentiation and exit of pluripotency. The lack of tumor formation at 6 weeks reported here indicates that double sorting is an effective approach at improving graft safety in the context of mESCs.
A factor that may have contributed to tumor formation in our single-sorted groups was the use of the immunocompromised mice. We observed that similar allografts in wild-type Sv129 hemiparkinsonian mice produced a much lower rate of tumor formation, with only 3 out of 46 animals exhibiting overgrowth. This indicates that there is a substantial risk of tumor formation, particularly when large numbers of mESC-derived neurons are grafted into the brains of immunocompromised mice. Future PD grafting studies will likely involve immunosuppression, suggesting that even under allograft conditions it will be important to proceed cautiously on the road toward future clinical translation of ESC-derived cell types.
A critical issue for DA neuron replacement paradigms in PD is the use of an appropriate cell source. While previous studies in mESCs have demonstrated in vivo functionality in murine PD models, there has been some concern that naive ESC-derived DA neurons may be less effective at alleviating behavioral symptoms as compared with fetal midbrain DA neurons. Several studies suggested that efficient DA neuron generation and engraftment occurs only upon transgenic modification of the cells using transcription factors such as NURR1 (25
), LMX1A (26
), or BCL-XL (27
). This study shows that DA neurons derived from naive ESCs achieve robust behavioral recovery. Therefore, the variable results obtained in previous studies using naive ESC sources may have been caused by a low percentage of authentic DA neurons, rather than reflecting a true requirement for the expression of transgenic DA neuronal fate determinants.
In this study, the authenticity of FACS-purified midbrain DA neurons was confirmed at the molecular level for both Nurr1::GFP+
expression in the developing mouse brain is not exclusive to the midbrain DA neuron lineage (28
). However, under the conditions of our differentiation protocol, Nurr1::GFP+
cells were consistent with the midbrain DA neuron phenotype based on both global transcriptome analysis and characterization of the grafted cells in vivo. Therefore, the incomplete marking of TH+
cells by FOXA2 upon replating Nurr1::GFP+
cells after sorting (Figure , B and J) likely reflects transient downregulation of the transcription factor, rather than alternative fate choice.
Gene discovery via the 3 reporter lines identified several relatively novel transcriptional regulators shown to be localized to the ventral midbrain, such as Foxp2
) and Ebf3
). The transcription factors, Uncx4.1
) and Pbx1
, were confirmed via qRT-PCR (Supplemental Figure 7A), with Uncx4.1
being enriched under both paradigms and Pbx1
being enriched only under the intraline analysis. Supporting this observation, Pbx1
expression was found throughout the E10.5 mouse midbrain (Supplemental Figure 7D) and had a much broader domain than UNCX4.1
in the developing human midbrain (Figure H), therefore suggesting a broader role for this transcription factor in CNS development. However, these genes continue to be expressed in the adult SN (Figure , A–D) and therefore are particularly attractive for future studies aimed at exploring the potential functional roles for these transcriptional regulators in midbrain DA neuron specification and/or maintenance. Additionally, the ability to purify midbrain DA neurons from hESC cultures will be necessary for any clinical translation, and therefore it is encouraging that CHRNA6 and CHRNB3 are cell surface markers expressed in the developing mouse and human midbrain, respectively (Supplemental Figure 9). Furthermore, a recent study has also confirmed the midbrain DA neuron specificity of the most significant cell surface marker gene on our gene list, Gucy2c
) (Table and Supplemental Tables 1–4). So while our data clearly demonstrate the applicability of SSRLA toward gene discovery, it would be interesting to compare our approach head-to-head with alternative approaches such as the use of transcription factor–driven lineage selection (33
) or FACS-based isolation of primary DA neurons.
In conclusion, our study emphasizes the importance of identifying the appropriate cell type and differentiation stage in achieving safe and efficacious DA neuron engraftment. We found that early postmitotic DA neurons at the Nurr1+
or later stage represent a promising cell source for future translational studies. The use of BAC transgenic reporter lines may not be suitable for human clinical translation due to the need for transgene insertion and reporter gene expression. However, our profiling studies present multiple candidate surface markers that may enable comparable antibody-mediated cell isolation strategies. The ultimate goal for translation may be, similar to that of drug-based therapies, to establish fully characterized, scalable, and ready-to-use populations of NURR1-stage DA neurons that can be properly dosed and standardized for therapeutic applications. While many hurdles remain on the road toward this goal, we recently described a novel strategy for deriving midbrain DA neurons from hESCs that resulted in excellent in vivo survival and function (34
). The choice of DA neuron differentiation stage used in this study was influenced by the current BAC reporter data, as we specifically aimed at transplanting hESC-derived DA neurons at the Nurr1
expression stage. However, formal comparisons using hESC-based BAC or knockin reporter lines will be required to formally address these questions in purified human cells. While DA neuron replacement in PD represents a symptomatic treatment unlikely to affect all disease-related symptoms, access to pure populations of engraftable DA neurons will be a major step forward toward testing the full potential of DA neuron replacement therapies in PD. Finally, we believe that SSRLA is a powerful application of ESCs for gene discovery that could be used for generating refined genetic signatures in other specific ESC-derived cell types.