The functional in vitro data using high sensitive ELISA sugg

The functional in vitro data using high-sensitive ELISA suggested that the 3D model-derived TH+ neurons secrete DA at a higher level than hESCs derived from the conventional 2D platform upon K+ induction. However, it is recommended that the ELISA data be validated via high performance liquid chromatography (HPLC) assay in the future. Also, further functional analysis is required such as electrophysiology using whole cell patch clamp in order to fully characterize the differentiated cells.
Conclusions In conclusion, the experiments herein demonstrated and provided as a proof-of-principle that 3D differentiation via microencapsulation is a powerful tool for DA neuronal differentiation. However, further analyses by differentiating the neurons under 3D and 2D platforms for longer period, followed by characterization of these ptio manufacturer using electrophysiology and transplantation assay in Parkinsonian rat model are required. Generating robust functional DA neurons efficiently is an essential requirement if cell therapy for PD is to become a reality. The study\'s proposed 3D platform is an effort in that direction.
Author contribution
Acknowledgments This work was funded by the National Health and Medical Research Council Program Grant (Perminder Sachdev) and Faculty of Medicine, University of New South Wales, Stem Cell Initiative.
Introduction Stem cells (SCs) that have the capacity to self-renew and to give rise to differentiated progeny are of broad interest because of their potential in regenerative therapy and their purported role in tumor initiation and relapse. The ability to identify and isolate SCs is essential for understanding molecular mechanisms underlying SC self-renewal and expansion, as well as their roles in tumorigenesis. Several approaches have been used to isolate or define SCs from a variety of organs. These include cell sorting based on SC surface markers (Stingl et al., 2006; Lawson et al., 2007; Spangrude et al., 1988), bromodeoxyuridine (BrdU) or other long-term label retention for slow cycling activities (Bickenbach and Chism, 1998; Tumbar et al., 2004), sphere-forming assays for the self-renewal property (Reynolds and Rietze, 2005; Dontu et al., 2003; Xin et al., 2007), and lineage tracing for their progeny (Snippert and Clevers, 2011). Emerging evidence shows that two types of SCs exist in various tissues, in separate yet adjoining locations. The slow-cycling SCs can be identified by long-term label retention and active SCs that do not retain labels due to rapid cell divisions can be identified by expression of Lgr5 (Leucine-rich repeat-containing G protein-coupled receptor5) (Snippert and Clevers, 2011; Barker et al., 2007, 2012; Huch et al., 2013). Teeth are highly mineralized organs derived from the dental epithelium and the underlying mesenchymal cells originally from neural crest, which undergo a series of sequential and tightly regulated processes to form a tooth (Grobstein, 1967; Thesleff and Hurmerinta, 1981; Thesleff et al., 1995). Five different lines of dental stromal cells that possess SC properties have been established from developing or mature human teeth (Gronthos et al., 2002; Miura et al., 2003; Sonoyama et al., 2008; Seo et al., 2004; Morsczeck et al., 2005). However, progressing in characterizing dental epithelial stem cells (DESCs) has been slow. It has been proposed that adult human teeth do not have DESCs since ameloblasts, the terminally differentiated dental epithelial cells, shed after tooth eruption in humans. Moreover, the lack of culture systems and well-accepted surface markers for DESCs further impede this research. Rodent incisors grow continuously throughout life, which is made possible by the existence of DESCs in the cervical loop (CL) region (Tummers and Thesleff, 2003; Yokohama-Tamaki et al., 2006; Harada et al., 1999; Harada and Ohshima, 2004). The presence of DESCs in the CL region is evidenced by the gradual differentiation of ameloblast-lineage cells apical to the incisal direction (Mitsiadis et al., 2007), the directional cell migration demonstrated by vital carbocyanine dye DiI tracking, long-term BrdU retention, cell cycle kinetics studies (Harada et al., 1999), and in vivo lineage trace of Sox2 expressing cells (Juuri et al., 2012).