The recombinant limb technique was developed to study osteo-chondrogenic differentiation and cartilage patterning using stem cells. The method is based on separating the ectoderm from the limb mesenchyme and refilling the ectodermal jacket with the material of interest. Then the engineered limb buds are either reinserted back into the lateral plate mesoderm of the developing chick or placed on the chorioallantoic membrane (CAM). The CAM is an extra-embryonic highly vascularized membrane that is formed from the infusion of mesodermal layers of the chorion and the allantois. Subsequently, tissue development is followed over several days.
We use the recombinant limb technique to successfully generate an organized structure from recombinant limbs of chicken mesenchymal cells and human IPS cell aggregates by incorporating a chicken ectodermal jacket. The mesenchymal stem cells were isolated from the limb bud mesenchyme of chicken embryos at HH stages 19-23 and then inserted into the chicken ectodermal jacket collected of the same stage embryos. For human-chick recombinant limbs, the undifferentiated human IPS cells were inserted in chicken ectodermal jacket stage 23 and the recombinant limbs were placed on the CAM.
The growing limbs were characterized using histological staining and in situ hybridization and immunochemistry methods to monitor the efficiency of the ectopic limb development.