Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

Abstract

Background: Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed.Methods: Monolayer cultured human embryonic stem cells and human induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and Alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. Results: The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrated the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. Conclusions: Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a step-wise induction system in the future.

Pluripotent stem cells for skeletal tissue engineering

“…Nonspecific differentiation as a monolayer (Figure 1(A)) is a simple strategy that involves the treatment of hPSC monolayers with a differentiation medium in order to generate a population of differentiated cells in a single step, without sequentially driving multiple stages of development. Promising hPSC differentiation protocols using this method are scarce, but in recent years protocols for osteogenesis have been reported [10,11]. One method involves the direct application of osteogenic medium [Minimum Essential Medium Eagle alpha modification, fetal bovine serum (FBS), nonessential amino acids, b-mercaptoethanol, ascorbic acid, sodium glycerophosphate and dexamethasone (DEX)] for 35 days.…”

“…One method involves the direct application of osteogenic medium [Minimum Essential Medium Eagle alpha modification, fetal bovine serum (FBS), nonessential amino acids, b-mercaptoethanol, ascorbic acid, sodium glycerophosphate and dexamethasone (DEX)] for 35 days. Despite enhanced expression of osteogenic markers, this strategy resulted in a highly heterogeneous population of cells, and low differentiation efficiency, evidencing the need for more specific methods [10]. A different strategy uses osteogenic medium supplemented with retinoic acid (RA), resulting in the formation of calcified osteogenic nodules, from which osteogenic cells can be isolated.…”

“…Strategies for nonspecific differentiation of hPSCs. (A) Nonspecific differentiation as a monolayer through treatment of PSCs with differentiation medium is an uncommon strategy but has been applied to osteogenic differentiation [10,11]. (B) Embryoid body (EB) formation enables spontaneous differentiation of the three embryonic germ layers.…”