Directed differentiation of human embryonic stem cells : a model for early bone development

Research in stem cell biology is an important and necessary requirement for the better understanding of cell differentiation and formation of tissues, while also contributing to the field of regenerative medicine.

The establishment of human embryonic stem cell (HESC) lines offers the potential to study the earliest developmental processes and provides an unlimited source of cells which can be used for the differentiation into functional osteoblasts. Bone matrix production and mineralization are guided by complicated mechanisms that differ from other tissues in many ways. There is the initial formation of an organic extracellular matrix (ECM) into which inorganic hydroxyapatite crystals are later deposited. Our first study investigated the molecular processes that occur pre- and post-mineralization within the primary ossification centre during early bone formation using global gene expression analysis. We then continued investigating the osteogenic differentiation potential of several HESC lines. Novel to our studies was the use of commercially available human foreskin fibroblasts to support the undifferentiated growth of the HESC colonies and their propagation in serum-replacement containing culture medium.

Two different approaches to differentiate HESCs into the osteogenic lineage were evaluated. Firstly, undifferentiated cells were cultured in suspension, facilitating the formation of embryoid bodies (EB), and secondly in monolayer; both methods were in the presence of osteogenic supplements. Characterization of the osteogenic phenotype revealed that all HESC lines differentiated towards the osteoblastic lineage, demonstrating also that EB formation is not necessary for the initiation of osteogenic differentiation. Mineralization of the ECM occurred through a cell-mediated calcification process. Study of the expression profile of bone-associated genes revealed that the HESC model differs from the standard osteogenesis model, which has been characterized by osteoprogenitor cells.

In the redefined model there is first the general cellular proliferation and secretion of pre-maturational matrix stage that is needed for cell migration, and second, the appearance of osteoprogenitors with characteristic ECM synthesis. A gene modification approach to enhance potential osteoblastic differentiation was employed in the fourth and final study. We found that for enhanced osteogenesis originating from in vitro cultured HESCs, the correct levels of ectopic transcription factors need to be established. Our data adds additional confirmation of a close relationship between early blood and bone development.

List of scientific papers

I. Sugars RV, Kärner E, Petersson U, Ganss B, Wendel M (2006). "Transcriptome analysis of fetal metatarsal long bones by microarray, as a model for endochondral bone formation." Biochim Biophys Acta 1763(10): 1031-9. Epub 2006 Aug 24
https://pubmed.ncbi.nlm.nih.gov/17005269

II. Kärner E, Unger C, Sloan AJ, Ahrlund-Richter L, Sugars RV, Wendel M (2007). "Bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro." Stem Cells Dev 16(1): 39-52
https://pubmed.ncbi.nlm.nih.gov/17233553

III. Kärner E, Bäckesjö CM, Cedervall J, Sugars RV, Ährlund-Richter L, Wendel M (2008). "Dynamics of gene expression during bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro." (Submitted)

IV. Kärner E, Unger C, Cerny R, Ährlund-Richter L, Ganss B, Dilber S, Wendel M (2008). "Authors have contributed equally to this study. Differentiation of human embryonic stem cells into osteogenic or hematopoietic lineages: a dose-dependent effect of Osterix over-expression." (Submitted)