Pluripotent joint progenitor cells, compositions and methods of making same
A technology of progenitor cells and pluripotency, applied in the field of pluripotent joint progenitor cells, their composition and preparation, can solve the problems of insufficient environmental clues for correct differentiation and repair of cells, and unsatisfactory results
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Embodiment 1
[0102] Example 1: Lgr5 as a new marker of distinct cells in developing synovial joints
[0103] As interstitial cells are progenitors, we screened these cells with a panel of stem cell markers and examined Lgr5 expression by q-RT-PCR ( Figure 9 ). Using GFP expression in Lgr5-eGFP-IRES-CreERT2 (Lgr5-GFP) mice, we identified Lgr5 as a marker for interstitial cells. Lgr5-GFP is a null allele in which GFP expression replaces Lgr5 (Barker et al., 2007). Heterozygous mice for this allele are normal and viable, whereas homozygous mice die perinatally (Barker et al., 2007). However, we did not observe abnormalities in limb development or synovial joint formation in homozygotes ( Figure 10 ). All analyses of Lgr5 / GFP expression in synovial joints were performed in mice heterozygous for this allele. The finger joints develop from proximal to distal, thereby providing information on progression. By whole-body embryo analysis of Lgr5-GFP mice, we detected GFP in knuckles from E13...
Embodiment 2
[0104] Example 2: Lgr5 expression starts after Gdf5 expression in knuckle formation
[0105] Gdf5 is a marker for interstitial cells (Merino et al., 1999; Storm and Kingsley, 1999). We compared adjacent slices in finger III ( figure 1 Expression of Lgr5 and Gdf5 in c-1f). Gdf5 is already expressed in the P2 / 3 interzone, the last joint formed at E14.5 ( figure 1 c), but not yet expressing Lgr5 ( figure 1 c), which indicates that the latter is expressed later. In the more proximal P1 / P2 and M / P1 joints, both Gdf5 and Lgr5 were expressed. Interestingly, Lgr5 expression was localized to the center-to-edge location of GDF5-expressing compartment cells in each joint ( figure 1 d). At E16.5, just before the formation of the articular cavity, Gdf5 was consistently expressed in the interspace region and exhibited a distinct horseshoe-shaped distribution ( figure 1 e), where Lgr5+ cells are located in the center of the horseshoe ( figure 1 f, circles), flanked by distinct Gdf5+ ...
Embodiment 3
[0106] Example 3: Expression of Lgr5 in the developing knee joint
[0107] The knee joint is more complex due to its additional structures with the meniscus and cruciate ligaments. Whole embryo imaging of E16.5 shows specific Lgr5 expression ( figure 1 g). We examined the periphery of developing joints from E13.5 to E18.5 ( figure 1 i) and central ( figure 1 j) Tissue sections of the area, such as figure 1 shown in h. Lgr5 is expressed in the interspace as early as E13.5, prior to the formation of the meniscus, articular cartilage and cruciate ligament. From E16.5, with early joint cavity formation and formation of the meniscus and cruciate ligaments, to E18.5 joint maturation, Lgr5 expression becomes localized to the future articular surface of the knee joint and attenuates ( figure 1 i; outer slices). However, at this stage, many Lgr5+ cells become evident in the developing lateral and medial menisci ( figure 1 i, outer slice). These Lgr5+ cells are located in the c...
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