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 integrate 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 stepwise induction system in the future.

Keywords: Human embryonic stem cells; Human-induced pluripotent stem cells; Marker expression; Osteogenic differentiation.

Fig. 1

Analyses of cell viability and telomerase activity for hPSCs during 35 days of osteogenic differentiation. a A schematic diagram of the experimental protocol. b, c After osteogenic induction for various days (0, 3, 7, 14, 21, 28 and 35), the cell viability of hESCs (b) and hiPSCs (c) was detected using the CCK8 reagent. d, e The telomerase activities of hESCs (d) and hiPSCs (e) were measured by a quantitative method based on a quartz crystal microbalance (QCM). *Represents p < 0.05 (n = 3)

Fig. 2

Analyses of the cell cycle for hPSCs during 35 days of osteogenic differentiation. a, b The cell cycle changes of the hESCs (a) and hiPSCs (b) after induction for different times (0 days, 3 days, 7 days, 14 days, 21 days, 28 days and 35 days) were studied using flow cytometry

Fig. 3

The expression of marker genes in hPSCs during osteogenic differentiation. a–g After osteogenic induction for up to 35 days, the expression of marker genes such as OCT-4 (a), NANOG (b), TERT (c), ALP (d), RUNX2 (e), COL1A1 (f) and OCN (g) in the cell samples was measured by RT-PCR. n = 3

Fig. 4

The expression of OCT-4 and RUNX2 in hPSC samples during osteogenic differentiation. The expression of OCT-4 (green) and RUNX2 (green) in the H9 hESCs and hNF-C1 hiPSCs after osteogenic induction for the indicated days were detected by immunofluorescence. The nucleus is shown in blue by DAPI staining. Scale bars, 100 μm

Fig. 5

The measurements for RUNX2-positive expression in hPSCs during osteogenic differentiation. After osteogenic induction for different days (0, 3, 7, 14, 21, 28 and 35), the expression of RUNX2 in the cells was measured by flow cytometry, and undifferentiated hPSCs were used as the control

Fig. 6

The expression of COL1A1 and OCN in the induced hESCs and hiPSCs. After osteogenic induction for 21 days, 28 days or 35 days, the expression of COL1A1 (red) and OCN (red) in the cell samples were detected by immunofluorescence. The nucleus is blue from DAPI staining. Scale bars,100 μm

Fig. 7

The Alizarin red staining analyses for the hPSCs during osteogenic differentiation. a, b Cell morphology and culture plate photograph of alizarin red staining of hESCs (a) and hiPSCs (b) after culturing in induction medium for up to 35 days. Scale bars, 200 μm. c, d Cetylpyridinium bromide solution was applied to dissolve the deposited alizarin red and the absorbance at 490 nm was measured. *Represents p < 0.05 (n = 3)

Fig. 8

A dynamic map of the osteogenic differentiation of the hPSCs. Expression changes of OCT-4, NANOG, TERT, ALP, RUNX2, COL1A1 and OCN and cell telomerase activity were investigated in the hPSCs during 35 days of osteogenic differentiation. The panels represent (from left to right) hPSCs that were induced for 0 days, 3 days, 7 days, 14 days, 21 days, 28 days or 35 days, which cover the various stages of osteoblastic lineage development