Abstract
Aim: To investigate the directed chondrogenic differentiation of human embryonic and adult stem cells in 3D alginate bead culture.
Introduction: Cartilage possesses limited self-renewal potential and current repair of damage due to trauma or disease involves removal of non-load bearing chon-drocytes from a healthy part of the joint, expansion of chondrocytes and subsequent surgery to replace damaged, load-bearing cartilage. We investigated the potential of human embryonic and adult stem cells as an alternative cell source for cartilage repair.
Experimental design: Human embryonic stem cells (hESC) and human adult marrow stromal cells (hMSCs) cells were cultured in alginate in a 3D bead format in control or chondrogenic media over a 21day period. Cells were subsequently released from their matrix for gene expression analysis or fixed within alginate beads and crytostat sections prepared for immunostaining and histology.
Cell types used: H9 human embryonic stem cells, bone-marrow derived hMSCs and HEK293 (human embryonic kidney epithelium cell line, used as a negative control).
Data: H9 and hMSC cells cultured in alginate beads bathed in control media have a denser matrix with no lacunae-like structures compared to those cultured in the presence of chondrogenic media. The presence of chondrogenic media results in a matrix containing cells within lacunae-like structures very similar to those seen in human cartilage. In contrast, HEK293 cells formed large highly cellular clusters which had clearly undergone significant proliferation. As both H9 and HEK293 cells are highly proliferative the reduction in the proliferative potential of the chondrogenic H9 derived cells is consistent with entry into a stable terminally differentiated state.
Immunostaining demonstrated that hMSCs and H9 cells express cartilage specific Collagen II and Collagen X.
Conclusion: 3D culture of adult hMSCs and hESC (H9) in alginate beads has resulted in stable directed differentiation down the chondrogenic lineage. These data point towards the future use of these human cell sources in cartilage repair.
Correspondence should be addressed to Mr Carlos Wigderowitz, Senior Lecturer, University Department of Orthopaedic and Trauma Surgery, Ninewells Hospital and Medical School, Dundee DD1 9SY.
