Neuroanatomical studies indicate that DARPP neurons could be

Neuroanatomical studies indicate that DARPP32+ neurons could be divided into two populations: one that constructs a direct pathway and another that constructs an indirect pathway. DARPP32+ neurons in the direct pathway express DRD1 and are located in the striosome. On the other hand, those in the indirect pathway express DRD2 and are located in the matrix (Gerfen et al., 1990; Crittenden and Graybiel, 2011). A recent neurotracing study indicated that DARPP32+ neurons in both the striosome and matrix are innervated by nigral DA neurons (Matsuda et al., 2009). Our study revealed that DRD1+ neurons are more frequently innervated by nigral DA neurons than are DRD2 + neurons. Interestingly, integrin α5 was dominantly expressed in the striosome, where the soma of DARPP32+ neurons and TH+ neuronal fibers had accumulated during the neonatal stage (Figure S3). These results suggested that the process of synapse formation between the grafted DA neurons and striatal neurons recapitulates the DA innervation process during development. In conclusion, we show that E2B could be used to activate integrin α5β1 in adult rodent striatum. Importantly, a qPCR analysis of postmortem human brains indicated that the expression levels of integrins α5 and β1 were still maintained in the putamen of PD patients (Figure 2). These results suggest that E2B could be used to modify the host bcl-2 inhibitor environment in a way that improves the outcome of cell transplantation therapy for PD.
Experimental Procedures
Author Contributions
Acknowledgments We thank Ms. Tomoka Ashida, Mrs. Yuko Ishii-Konoshima, Mr. Kei Kubota, and Mr. Yoshifumi Miyawaki for technical assistance. We also thank other laboratory members for helpful discussions, Dr. Yuichi Ono (KAN Research Institute) for providing anti-CORIN antibody and anti-NURR1 antibody, Dr. Ryoma Morigaki (University of Tokushima) for valuable comments, and Dr. Peter Karagiannis for critical reading of the manuscript. We also thank professor Ernest Arenas (Karolinska Institute) for helpful comments. This research was supported by the Ichiro Kanehara Foundation (K.N.), a Grant-in-Aid for Young Scientists (B) to K.N. (26861146), and the Network Program for Realization of Regenerative Medicine (J.T.) from the Japan Science and Technology Agency, and an Intramural Research Grant for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry to J.T. (24-9). B.S. is a recipient of a JSPS DC2 fellowship (14J02729).
Introduction Pluripotent embryonic stem cells (PSCs) facilitate research on mammalian neuronal development, neurodegenerative disorders, and regenerative therapies. It has been shown in the retina that developmental processes such as optic-vesicle (OV) and optic-cup (OC) morphogenesis and signaling cascades can be reproduced using mouse and human embryonic stem cells (mESCs and hESCs) (Eiraku et al., 2011; Nakano et al., 2012; Hiler et al., 2015; La Torre et al., 2015). Retinal organoid (Boucherie et al., 2013; Decembrini et al., 2014; Gonzalez-Cordero et al., 2013) and 2D culture approaches (Lamba et al., 2006; Osakada et al., 2008) have been used for cell replacement therapy studies because efficient derivation of sufficient numbers of integration-competent cells remains a major limitation for regenerative medicine. The first reports on cell-based disease-modeling approaches (Phillips et al., 2014), retinal neuronal morphogenesis (Busskamp et al., 2014), and function in organoids (Zhong et al., 2014) are promising. Yet, in this evolving field, benefits and limitations have not been fully explored and many questions remain. For example, the question of efficient generation of large, stratified, retinal tissues has not been addressed. Sasai and colleagues pioneered a protocol that allows the self-organization of eyecup-like structures (Eiraku et al., 2011; Nakano et al., 2012). This entails a series of complex tissue interactions, such as eyefield evagination and subsequent invagination, resulting in neural retina opposed by retinal pigment epithelium (RPE). However, this protocol relies on the evagination of the neuroepithelium and its live visualization, preferably using transgenic RAX (retina and anterior neural fold homeobox) reporter gene expression, for reliable manual isolation of the prospective retinal organoids. RAX is part of a group of transcription factors sufficient and necessary for the specification of the eyefield, which gives rise to the eye primordia and the retina. Although eyefield formation has been shown to be efficient in mouse PSC lines, the yield of retinal organoids depends on and is highly limited by a low frequency of neuroepithelial evagination (Eiraku et al., 2011; Hiler et al., 2015). Others have adapted protocols to maximize and simplify rod photoreceptor production by omitting the evagination dissection step. This results in larger organoids, with retinal and non-retinal structures intertwined within the starting organoid, and comes at the expense of inner-retina cell types (Decembrini et al., 2014; Gonzalez-Cordero et al., 2013). Therefore, we speculated that unbiased neuroepithelium trisection at the eyefield stage overcomes these limitations and enables production of more numerous retinal organoids.