During gastrulation the first mesodermal cells including hem

During gastrulation, the first mesodermal Phenyl sulfate including hematopoietic progenitors emerge from the posterior part of the early primitive streak migrating anteriorly and laterally. Cardiac, cranial, and lateral plate mesoderm progenitors are formed during the mid-streak stage, followed by the ingression of progenitors that specify trunk and paraxial mesoderm (muscle, bone, and cartilage precursors) in the late-streak stage (Loebel et al., 2003; Kinder et al., 2001). Cartilage formation during endochondral bone development is initiated by mesenchymal condensation, within which cells differentiate into chondrocytes that express the transcription factor SRY (sex-determining region Y)-box 9 (Sox9) (Lefebvre et al., 1997; Bi et al., 1999; Akiyama et al., 2002; Lefebvre and Smits, 2005), which regulates the production of type II collagen (Col2a1) and the proteoglycan Aggrecan (Goldring et al., 2006; Poole et al., 2001). Proteoglycan-4 (PRG4) encodes a superficial zone protein synthesized specifically by articular cartilage (Schumacher et al., 1999; Flannery et al., 1999). Chondrocytes undergo hypertrophy prior to matrix calcification (Olsen et al., 2000; Provot and Schipani, 2005; de Crombrugghe et al., 2001), producing type X collagen (Col10a) and expressing the master regulator of osteoblast differentiation runt-related transcription factor 2 (Runx2) (Karsenty and Wagner, 2002; Otto et al., 1997; Komori et al., 1997). ESC models of primitive streak formation and lineage commitment validate the possibility of generating functional cell types under defined conditions (Gadue et al., 2006; Kattman et al., 2006; Nostro et al., 2008; Murry and Keller, 2008). The majority of in vitro chondrogenic differentiation strategies rely on the establishment of dense pellet or micromass cultures in serum-containing or conditioned media to mimic mesenchymal condensation. However, the 3D clustering of heterogeneous cell populations creates an unknown culture environment that obscures the effects of exogenous factors due to the increased occurrence of fate-determining paracrine interactions among various cell populations, in comparison with 2D cultures, while the presence of serum components masks the effects of growth factors.
Results
Discussion We examined if long-term treatment of murine ESC SF monolayer cultures with BMP4, activin A, Wnt3a, TGFβ3, and FGF8 at the onset of differentiation could direct chondrogenic differentiation with minimal culture manipulation. Activin A, BMP4, and TGFβ3 acted as early inducers of chondrogenesis in our culture system while Wnt3a exerted its prochondrogenic effect only after mesoderm specification (Fig. 7). ECM selection was crucial as 2-day LIF-supplemented SF ESC cultures established on gelatin+fibronectin exhibited accelerated differentiation with 5–10% lower OCT4 levels compared to cultures on gelatin or collagen IV (Figs. 1A and B). The bimodal OCT4 expression profile was commonly observed in other HCI assays established on gelatin+fibronectin (Davey and Zandstra, 2006; Walker et al., 2007). Although fibronectin is endogenously expressed by differentiating ESCs (Hayashi et al., 2007) and promotes cell adhesion and spreading (Dufour et al., 1986), we rejected gelatin+fibronectin for our system to minimize spontaneous ESC differentiation toward undesired lineages in the absence of inductive factors. Gelatin, being a mixture of collagens, was less defined than collagen IV, and collagen IV has been shown to facilitate ESC differentiation toward the mesodermal lineages (Nishikawa et al., 1998; Tada et al., 2005; Sakurai et al., 2006). Despite the successful establishment of adherent chondrocyte cultures on fibronectin or collagen (Khan et al., 2009; Ho et al., 2009), gelatin+fibronectin cultures did not show enhanced Brachyury or chondrogenic markers expression compared to collagen IV cultures (data not shown). Similar to published EB studies (Nostro et al., 2008), BMP4, activin A, and Wnt3a all induced Flk1 (Figs. 2B and C) and Brachyury expression (Figs. 3A and B) in our 4-day monolayer differentiation cultures. Activin A- or Wnt3a-treated cultures consisted of flattened colonies with stronger adhesion and spreading on collagen IV than with BMP4 (Fig. 2A). Conversely, there was ~6–8% more of annexin V+ apoptotic cells present in BMP4 cultures than in Wnt3a and activin A cultures (Fig. 2C). Activation of the TGFβ signaling pathway induces epithelial-to-mesenchymal transition (EMT), leading to the up-regulation of neural cell adhesion molecule (NCAM) (Thiery and Sleeman, 2006). NCAM promotes the phosphorylation of focal adhesion kinase (FAK) and integrin-dependent cell spreading (Frame and Inman, 2008). FAK phosphorylation alters its downstream target Grb2 and facilitates its interaction with the Ras/mitogen-activated protein kinase (MAPK) pathway, which modulates cell survival and proliferation (Schlaepfer et al., 1994; Harburger and Calderwood, 2009). Members of the canonical Wnt and integrin signaling pathways (specifically the collagen-binding integrins α1β1 and α2β1) have been shown to act synergistically via Grb2, (Crampton et al., 2009), possibly contributing to the satisfactory cell spreading and survival observed in Wnt3a-supplemented cultures. The non-uniform morphology of differentiating colonies in BMP4 culture (Fig. 2A, iv) could be due to the potency of BMP4 at 10ng/ml as the cultures also showed weaker Brachyury protein expression (Fig. 3A, iv); consequently, serum-deprivation promoted apoptosis in the slowly differentiating cells. Alternatively, although BMP4 (10ng/ml) was less potent than activin A or Wnt3a in EMT initiation and mesoderm induction, its presence was sufficient to prevent neuroectoderm differentiation in our culture system by inducing apoptosis in early precursors of neural cells (Gambaro et al., 2006).