Commitment of human mesenchymal stromal cells to skeletal lineages is independent of their morphogenetic capacity

Figure 1 Validation of the recombinant limb system using human mesenchymal stromal cells. A: Implantation efficiency of umbilical cord blood (UCB)-recombinant limb (RLs) and placenta (PL)-RLs in chicken embryos after 24 h. Scanning electron microscopy of PL-RLs (n = 3) and UCB-RLs (n = 2) at 24 h postimplantation (hpi). The embryonic ectoderm and apical ectodermal ridge were conserved in RLs from both sources; B: Levels of human leukocyte antigen (Hla) detected by quantitative polymerase chain reaction at 24 hpi RLs from PL and UCB relative to its own pellet. Data represent three independent experiments. The major histocompatibility complex (HLA) expression pattern is shown in RLs from PL (n = 3) and UCB (n = 2). HLA expression was observed in the center of the RLs, indicating that mesenchymal stromal cells constituted the RLs. Statistical significance was set as follows: ^aP < 0.05; ^cP < 0.0005. UCB: Umbilical cord blood; PL: Placenta; RL: Recombinant limb; HLA: Human leukocyte antigen.

Figure 2 Phenotypic and histological characterization of the mesenchymal stromal cells-recombinant limbs. Alcian blue and hematoxylin and eosin staining of recombinant limbs from the placenta (n = 8), umbilical cord blood (n = 7), and chicken (n = 4) at 24, 48, and 72 h postimplantation. hpi: Hour postimplantation; UCB: Umbilical cord blood; PL: Placenta; RL: Recombinant limb; CK: Chicken; H&E: Hematoxylin and eosin.

Figure 3 Mesenchymal stromal cells differentially commit to chondrogenic, osteogenic, and tenogenic lineages in the recombinant limb system. A: Quantitative polymerase chain reaction analysis of cartilage (a-e), bone (f), and tendon (g and h) cell fate-regulating genes of the mesenchymal stromal cells-recombinant limbs (MSCs-RLs) at 24 h postimplantation (hpi) relative to its own pellet set to 1.0. Statistical significance comparing MSCs-RLs from the two sources is shown; B: Protein expression pattern of master genes of the three differentiation lineages in MSCs-RLs at 24 hpi. Data represent three independent experiments. Statistical significance was set as follows: ^aP < 0.05; ^bP < 0.005; ^cP < 0.0005; ^dP < 0.0001. For more details see Table 1. Sox9: SRY-box transcription factor 9; Gdf5: Growth differentiation 5; Col2α1: Collagen type 2 alpha 1; Acan: Aggecan; Hif1α: Hypoxia-inducible factor 1 alpha; Runx2: Runt-related transcription factor 2; Scx: Scleraxis; Mkx: Mohawk; UCB: Umbilical cord blood; RL: Recombinant limb; PL: Placenta; NS: Not significant.

Figure 4 Cell death and fibroblast growth factor 8 expression pattern in mesenchymal stromal cells and chicken recombinant limbs at 24 h postimplantation. Lysotracker staining (Ltk) to evaluate cell death in recombinant limbs (RLs) from the placenta (PL-RLs; n = 6), umbilical cord blood (UCB-RLs; n = 6), and chicken (CK-RLs; n = 4) is shown. In situ hybridization of fibroblast growth factor 8 (Fgf8) in PL-RLs showed disrupted pattern of this gene in 2/4 RLs; Fgf8 expression in UCB-RLs was detected in 3/6 evaluated samples, while 5/5 CK-RLs continued to express Fgf-8 at 24 h postimplantation. UCB: Umbilical cord blood; PL: Placenta; RL: Recombinant limb; CK: Chicken; Ltk: Lysotracker staining; Fgf8: Fibroblast growth factor 8.

Figure 5 Mesenchymal stromal cells commit to limb mesodermal lineages but are not capable of forming skeletal structures. Mesenchymal stromal cells (MSCs) from umbilical cord blood (UCB) and placenta (PL) differentiated into chondrogenic, osteogenic, and tenogenic lineages in vitro. However, under the influence of differentiation and morphogenetic signals from limb ectoderm in the recombinant limb (RL) system, MSCs committed to limb lineages but failed to organize into tridimensional structures and form skeletal elements as observed with limb bud mesodermal cells under the same conditions. Similarly, the response to ectodermal signals depended on the source of MSCs: UCB-RLs had higher expression of osteogenic and tenogenic markers than PL-RLs, whereas in PL-RLs, tenogenic markers were downregulated after receiving ectodermal signals. UCB: Umbilical cord blood; PL: Placenta; LBMC: Limb bud mesodermal cell; RL: Recombinant limb.