3D osteogenic differentiation of human iPSCs reveals the role of TGFβ signal in the transition from progenitors to osteoblasts and osteoblasts to osteocytes

Abstract

Although the formation of bone-like nodules is regarded as the differentiation process from stem cells to osteogenic cells, including osteoblasts and osteocytes, the precise biological events during nodule formation are unknown. Here we performed the osteogenic induction of human induced pluripotent stem cells using a three-dimensional (3D) culture system using type I collagen gel and a rapid induction method with retinoic acid. Confocal and time-lapse imaging revealed the osteogenic differentiation was initiated with vigorous focal proliferation followed by aggregation, from which cells invaded the gel. Invading cells changed their morphology and expressed osteocyte marker genes, suggesting the transition from osteoblasts to osteocytes. Single-cell RNA sequencing analysis revealed that 3D culture-induced cells with features of periosteal skeletal stem cells, some of which expressed TGFβ-regulated osteoblast-related molecules. The role of TGFβ signal was further analyzed in the transition from osteoblasts to osteocytes, which revealed that modulation of the TGFβ signal changed the morphology and motility of cells isolated from the 3D culture, suggesting that the TGFβ signal maintains the osteoblastic phenotype and the transition into osteocytes requires down-regulation of the TGFβ signal.

Figure 1

Induction of osteoblastic and osteocytic cells from hiPSCs (414C2) on type I collagen gel. (a) Histological findings of vertical sections of induced cells on type I collagen gel at days 7, 10 and 14 by Hematoxylin–Eosin (HE) staining. (b) Histological findings of a horizontal section at day 14 by HE staining. (c) Histological findings of a vertical section at day 14 with immunostaining for DMP1 (green) and DAPI (blue). (d) Histological findings of a horizontal section at day 14 with immunostaining for DMP1 (green), type I collagen (red) and DAPI (blue). Arrowheads show cells surrounded by COL I. Scale bars = 100 μm.

Figure 2

Dynamic behavior of hiPSCs during osteogenic induction. Horizontal (a) and vertical (b) snapshots of time-lapse imaging during the osteogenic induction of GFP-labeled hiPSCs (W2G) on type I collagen gel. Images were taken at the indicated times. Scale bars = 100 μm in (a) and X, Y: 508 μm and Z: 225 μm in (b). Confocal images of hiPSCs (404C2) in vertical (c) and horizontal (d) views during the osteogenic induction. Sections were stained with Phalloidin (green) and anti-COL 1 antibody (red). Arrowheads show cells surrounded by COL I.

Figure 3

Expression of stage-related markers in 3D culture. (a) Reconstructed 3D view of confocal images at day 14. Collagen gels with induced cells (414C2) were stained with antibodies for PHEX (green) and OCN (red), and immunofluorescence images obtained by confocal analysis were reconstructed to build 3D images. (b–g) Immunofluorescence analysis by confocal imaging. Collagen gel with induced cells at day 14 were stained with Phalloidin (magenda) and DAPI (skyblue) (b,d,f) or antibodies for PHEX (green) and OCN (red) (c,e,g). Horizontal images were analyzed at surface (d,e) and deep (f,g) levels indicated by the white and red lines in (b) and (c), respectively. Scale bars = 50 μm.

Figure 4

Single-cell expression profiles of osteogenic-induced cells. (a) UMAP plot of cell-type clusters. Cells from whole cell fraction (WC) and invading cell fraction (IC) were combined and analyzed and clustered into 15 subsets. (b) Relative contribution of WC- and IC-derived cells in each cluster. (c) Pseudotime trajectory analysis demonstrated the differentiation direction from cells colored in purple to those in yellow. (d) Average expression and percent expression of representative stage-related genes in each cluster with dot plots. (e) Visualization of cells expressing representative stage-related genes in (d) by UMAP.

Figure 5

Cell invasion required the activity of MMP. Histological findings of osteogenic induced hiPSCs (414C2) on type I collagen gel at day 14. Cells cultured with collagen gel were stained with Hematoxylin–Eosin (HE) (a), antibodies for p-Smad3 (green) (b), MMP2 (green) and DAPI (blue) (c,d), or MMP14 (green) and DAPI (blue) (e,f). Images from vertical (a,b,c,e) and horizontal (d,f) sections are shown. Scale bars = 100 μm. Histological findings of osteogenically induced hiPSCs (g–j) or MLO-Y4 (k,l) at day 14 in vertical sections by HE staining. Cells were treated without (g,h,k) or with (i,j,l) GM6001 (25 μM). (h,j) are enlarged views of the red-framed areas in (g) and (h), respectively. Arrowheads in (g) indicate invading cells. Scale bars = 1 mm.

Figure 6

Effect of TGFβ signal on osteogenic-induced iPSCs on type I collagen gel. The whole cell fraction of osteogenic-induced hiPSCs (414C2) was isolated at day 14 and cultured for 48 h in control medium (a,d,g) or medium supplemented with TGFβ1 (5 ng/mL) (b,e,h) or SB431542 (10 µM) (c,f,i). Cells were stained with Phalloidin (red) and DAPI (blue) (a–c) or antibody for COL I (d–f). (g–i) Merged images. Snapshots of the time-lapse imaging of isolated cells from days 1 to 3 cultured in control medium (j) or medium supplemented with SB431542 (10 µM) (k). Scale bars = 100 μm.