Bone Matrix Formation in Osteogenic Cultures Derived from Human Embryonic Stem Cells in Vitro

Abstract

Bone matrix production and mineralization involves sophisticated mechanisms, including the initial formation of an organic extracellular matrix into which inorganic hydroxyapatite crystals are later deposited. Human embryonic stem (hES) cells offer a potential to study early developmental processes and provide an unlimited source of cells. In this study, four different hES cell lines were used, and two different approaches to differentiate hES cells into the osteogenic lineage were taken. Undifferentiated cells were cultured either in suspension, facilitating the formation of embryoid bodies (EBs), or in monolayer, and both methods were in the presence of osteogenic supplements. Novel to our osteogenic differentiation study was the use of commercially available human foreskin fibroblasts to support the undifferentiated growth of the hES cell colonies, and their propagation in serum replacement-containing medium. Characterization of the osteogenic phenotype revealed that all hES cell lines differentiated toward the mesenchymal lineage, because T-Brachyury, Flt-1, and bone morphogenetic protein-4 could be detected. Main osteoblastic marker genes Runx2, osterix, bone sialoprotein, and osteocalcin were up-regulated. Alizarin Red S staining demonstrated the formation of bone-like nodules, and bone sialoprotein and osteocalcin were localized to these foci by immunohistochemistry. Cells differentiated in monolayer conditions exhibited greater osteogenic potential compared to those from EB-derived cells. We conclude that in vitro hES cells can produce a mineralized matrix possessing all the major bone markers, the differentiation of pluripotent hES cells to an osteogenic lineage does not require initiation via EB formation, and that lineage potential is not dependent on the mode of differentiation induction but on a cell line itself.

Phenotypic Characterization, Osteoblastic Differentiation, and Bone Regeneration Capacity of Human Embryonic Stem Cell–Derived Mesenchymal Stem Cells

“…3D) in hESCs-MSCs cultured in MSC medium and Runx2 in hESCs-MSCs cultured in osteogenic medium (Fig. 3K, 5) suggest that hESCs differentiated to mesenchymal stem cells and subsequently differentiated into osteoblastic cells (6,33,46). The sequential pattern of differentiation of hESCs-MSCs into mature osteoblasts (Fig.…”

“…Here, we did not introduce the hESCs to osteogenic medium at the EB stage, rather, the implanted cells were enriched osteoprogenitor cells derived from serial expansion of aggregates of hESCs in culture medium supplemented with FBS (6–9, 11). We then further cultured the hESCs-MSCs in osteogenic medium and transplanted a FAC-sorted population in vivo.…”

“…The advantage of ALP selective cell sorting is to enrich the numbers of ALP-positive colonies (55) and to obtain an enriched population of pre-osteoblasts in the late proliferation stage (Stro-1−/ALP+) (33). However, it is possible that ALP sorted cells may be contaminated with pluripotent cells, as ALP is expressed in undifferentiated hESCs (6), and is also expressed in cultured pre-osteoblastic and osteogenic cells (33,55). To determine the presence of undifferentiated hESCs or pluripotent cells in the osteogenically differentiated cells, RT-PCR was performed to compare expression of key regulators of pluripotency, Oct4 and Nanog genes (56) and the osteoblast-related genes, Runx2, collagen type I and bone sialoprotein (33,57) in undifferentiated hESCs, differentiated hESCs-MSCs in osteogenic medium and the enriched population of ALP expressing cells for transplantation (Figure 5).…”

Induced pluripotent stem cells from patients with human fibrodysplasia ossificans progressiva show increased mineralization and cartilage formation

“…Primary human MSCs were cultured in OB mineralization medium (DMEM with 20% FBS, glycerol-2-phosphate, 4 μM dexamethasone, 0.1 mM 2-mercaptoethanol, and 50 μg/ml L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate [12]). We could detect mineralization activity after 12 days as increased black staining by von Kossa (Additional file 2: Figure S1B), which was the preferred staining method used because the black mineralization nodules could be easily distinguished from the expected light golden-yellow staining of the cell layer.…”

Engineering the human pluripotent stem cell microenvironment to direct cell fate

“…These include monolayer, EB, and hybrid approaches. To demonstrate the 3D vs. 2D extremes, hESCs underwent osteogenesis in either an EB or monolayer system (Karner et al, 2007). While cells differentiated via both methods produced mineralized matrix, the monolayer cells had greater osteogenic potential.…”