In addition to these benefits growing SFEBs

In addition to these benefits, growing SFEBs on MIs has similar advantages to organotypic slice culture preparations. Brain slice cultures allow maximal flexibility when performing both electrophysiological and imaging experiments (Thompson et al., 2006). The protocol outlined here is particularly useful in live-cell studies that involve the tracking of iPS cell dynamics over periods of time within a 3-D system. SFEBs prepared using our approach allow high spatial resolution to be achieved in a live-cell setting using standard epifluorescence microscopy, as opposed to requiring two-photon laser scanning microscopy for thicker preparations.
Acknowledgments We thank Dr. Kevin Eggan, Dr. Lee Rubin, Dr. Steve Chang and Dr. Scott Lipnick for comments and suggestions. We also thank Chris Woodard and Seong Im Hong for help with cell quantification. This work is generously supported by grants from the Charles Evans Foundation, Alzheimer\'s Drug Discovery Foundation, and NY Community Trust.
Introduction Cartilage is a connective tissue formed by only one cell type, the chondrocyte, trapped in the extracellular matrix composed mainly of collagen fibers, aggrecan (a large aggregating proteoglycan) and water. In fact, many efforts are currently being made to create cartilage in laboratories by combining novel biomaterials, growth factors and amyloid (Moreira-Teixeira et al., 2011). Clinical treatment for articular cartilage injury includes autologous cell injections of primary chondrocytes expanded in vitro. However, the dedifferentiation of the autologous chondrocytes in culture and the small number of cells that can be obtained limit their clinical applications only to small injuries (Schuurman et al., 2012). Alternative cell sources for cartilage tissue engineering are mainly embryonic stem cells and adult stem cells. Mesenchymal stem cells derived from bone-marrow (MSCs) and adipose tissue (ASCs) have shown significant chondrogenic potential (Diekman et al., 2010; Estes et al., 2010; Pittenger et al., 1999). Importantly, ASCs can be easily harvested, expanded in vitro and are relatively abundant in comparison with MSC. Therefore, ASCs could be an ideal cell type for generating a large number of functional chondrocytes. The transforming growth factor-β (TGF-β) superfamily is comprised of almost forty ligands responsible for numerous cellular processes including early embryonic development, tissue patterning and homeostasis, bone formation, wound healing and fibrosis (Attisano et al., 1993; Hogan, 1996a, 1996b). These proteins signal through the simultaneous interaction with one of the 7 type I and one of the 5 type II TGF-β receptor (TGFR-β) kinases. The signaling requires the hetero-dimerization and subsequent activation of both types I and II receptors through the binding of their TGF-β ligands (Derynck and Feng, 1997). Interestingly, different TGF-β ligands, such as Activin (Jiang et al., 1993) as well as several isoforms of bone morphogenetic protein (BMPs) (Denker et al., 1999; Estes et al., 2010; Kramer et al., 2000; Majumdar et al., 2001), TGF-β1 (Awad et al., 2003; Erickson et al., 2002) or TGF-β3 (Hennig et al., 2007), have been shown to promote chondrogenesis. Since Activin A exhibits high affinity for type II receptors and signals through SMAD2/3 transcription factors, while BMP2 possesses higher affinity for type I receptors and signals through SMAD1/5/8 transcription factors, we investigated whether AB2 chimeras created by mixing Activin and BMP2 sequences (Allendorph et al., 2011) would be useful in promoting chondrogenesis. In short, BMP2 and Activin A sequences have been divided into 6 structural segments and these segments have been mixed to create the AB2 library of chimeras with novel functional properties. A systematic swapping strategy of the segments termed Random Assembly of Segmental Chimera and Heteromers (RASCH) was described in detail in Allendorph et al. (2011). The chimeras are fully defined by the code (BXXXXX), where X=A (Activin A) or B (BMP2) depending on which segment is in position X.