br Results br Discussion We first identified surface markers

Results
Discussion We first identified surface markers that distinguish the earliest prechondrocytes from nonchondrogenic sphingosine 1-phosphate receptor modulator types; subsequent comparison of prechondrocytes with differentiated, definitive resting (immature) chondrocytes located in the superficial periarticular regions within fetal joints led to the identification of a combination of surface markers enriched on both cell types and demarcating them from chondrocytes undergoing maturation and hypertrophy. The expression of CD166 and CD146 has been previously reported to mark highly invasive and migratory mesenchymal cells in normal and pathological conditions (Swart, 2002; Swart et al., 2005; Zeng et al., 2012). Moreover, CD146 has been shown to be directly involved in the process of epithelial-to-mesenchymal transition through the positive regulation of Slug (Zeng et al., 2012). In good agreement with these studies, loss of the mesenchymal phenotype and transition of cartilage-committed mesenchymal cells or prechondrocytes to differentiated nonmotile chondrocytes is associated with the progressive loss of CD166 of CD146. A combination of TGF-β1 and LIF was used in this study to inhibit excessive cartilage maturation and hypertrophy and favor the generation and persistence of primary and PSC-derived cartilage progenitors. Although the antidifferentiation properties of LIF have been well documented in the fields of pluripotent stem cells, hematopoiesis, and neurobiology (Audet et al., 2001; Bonaguidi et al., 2005; Smith et al., 1988), their role in the regulation of chondrogenesis is completely novel. Synovial cells of both fetal and adult normal joints highly express LIF (Figure 5), indicating that this factor may be implicated in the maintenance of the most primitive progenitors located in the superficial layer of articular cartilage. Indeed, LIFR was primarily localized in resting chondrocytes also positive for BMPR1B, while LIF and TGF-β1 and TGF-β2 were among the top molecules enriched in definitive resting chondrocytes (Table 1). Moreover, our validation studies on fetal chondrocytes suggest that these factors may prevent chondrocyte maturation and hypertrophy. More studies are needed to characterize BMPR1B+ cells in adult joints and fully define the role of LIF in the regulation of these cells. Our data suggest that utilizing the LIF signaling axis may further advance engineering of functionally superior cartilage implants for joint surface repair in patients with cartilage injury or arthritis. In summary, we have for the first time characterized the earliest stages of human chondrogenic development. Starting with the distinct morphological identity of prechondrocytes, we found a unique combination of surface markers (CD166low/negCD146low/negCD73+CD44lowBMPR1B+) that distinguishes these cells from other populations in the developing limb and directs the enrichment of chondrogenic progenitors. Subsequent comparison of the gene expression profile of prechondrocytes with resting periarticular chondrocytes, in conjunction with immunohistochemical analysis, led to the identification of the LIF, TGF-β, and BMP signaling pathways as potential regulators of the differentiation state of chondrocytes. Application of these findings permitted the generation of functional immature chondrocyte cells from pluripotent stem cells. The identification of specific phenotypic signatures for primitive prechondrocytes and definitive resting chondrocytes will now permit further delineation of the molecular regulation of chondrogenic commitment, growth plate versus articular cartilage fate choice, and hypertrophy in humans. It may also contribute to understanding of how these processes are affected during aberrant chondrogenesis in disease states. Finally, characterization of the earliest primary chondrocytes provides essential knowledge for the generation of purified cartilage cells from PSCs and represents a unique target for cartilage tissue engineering.