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


Induction of osteoblastic and osteocytic cells on type I collagen gel

The osteogenic induction of hiPSCs (414C2) was performed on type I collagen gel using a previously reported method on matrigel-coated dishes17, and the differentiation process was observed using histological specimens. Vertical sections at each time point during the induction revealed the gradual increase of cells in the gel (Figs. 1a and S1a). Horizontal sections on day 14 demonstrated that the surface of the gel was covered by a sheet-like structure of cuboidal cells and cells inside of the gel showed a dendritic morphology connecting with adjacent cells (Figs. 1b and S1b). Immunostaining of a vertical section showed that cells inside of gels were positive for DMP1 (Fig. 1c,d) and that of a horizontal section showed a mesh-like distribution of human type I collagen (COL I) beneath the layer of cuboidal cells (Figs. 1d and S1c).

Figure 1
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.

To compare the characteristics of these cells with those of established mouse cell lines, osteoblastic (MC3T3-E1) and osteocytic (MLO-Y4) cells18 were seeded on the collagen gel and cultured with the osteogenic induction method. In the case of MC3T3-E1, vertical sections at day 14 showed few cells in the gel (Fig. S2a), whereas many MLO-Y4 cells were found in the deep area of the gel (Fig. S2d). Horizontal sections showed multilayers of MC3T3-E1 cells on the surface (Fig. S2b), and a newly formed mesh-like Col I matrix was found beneath the cell layer (Fig. S2c). On the other hand, many MLO-Y4 cells were found in the gel (Fig. S2e), in which little Col I matrix was found (Fig. S2f.). These findings were comparable with those of osteogenic-induced hiPSCs, suggesting that the 3D culture system successfully induced osteoblastic and osteocytic cells from hiPSCs.

Dynamic behavior of hiPSCs during osteogenic induction

As demonstrated in our previous study17, the dynamic behavior of hiPSCs during osteogenic induction on type I collagen gel was visualized by time-lapse imaging using GFP-labeled iPSCs. Using the same imaging, horizontal images showed that induced cells became confluent at day 4 and focal cell aggregation appeared around day 10, gradually enlarged and produced a condensed nodule at day 14 (Fig. 2a and Mov. S1). In the vertical images, cells from the aggregates beginning day 7 further invaded the deep area and developed a dendritic morphology at day 10. At day 14, the aggregated cells sank into the gel as a mass, from which many cells actively invaded the deep area and showed dendritic morphology (Figs. 2b and Mov. S2).

Figure 2
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.

To investigate the process in detail, 414C2-derived cells with collagen gel were stained by Phalloidin and an antibody for COL I at different time points and analyzed by confocal microscopy. Vertical views showed COL I beneath the surface cell layer as early as day 2, and the expression of which gradually increased and mixed with pre-existing collagens from day 4 to day 7 (Figs. 2c and S3a). At day 7, focal cell proliferation began. The proliferating cells embedded into the gel as a mass at day 10, and some separated from the mass to invade the deeper area (Fig. 2c). Horizontal sections at day 14 were analyzed at several levels (Figs. 2d and S3b). The uppermost section shows a sheet-like structure of cuboidal cells, and the middle section shows cells surrounded by or burred in COL I matrix (Figs. 2d and S3b, indicated by arrowheads). The lowest section shows dendritic cells and little production of COL I. These results indicated that hiPSCs differentiated into osteoblastic and osteocytic cells through nodule formation and invasion into collagen gel.

Location-dependent expression of stage-related markers by cells in 3D culture

To further characterize cells during the transition process, the expression of stage-specific markers at day 14 was visualized by immunohistochemical staining and confocal imaging. A reconstructed 3D view clearly showed differences between cells on the surface and in the gel (Fig. 3a). Cells on the surface were positive for osteocalcin (OCN), whereas cells positive for PHEX were found mostly in the gel (Fig. 3a). **Horizontal sections at different depths were further analyzed (Figs. 3b,c, and S4a). In the surface layer, cells making a sheet-like structure were positive for OCN (Fig. 3d), and some cells around nodules expressed PHEX (Figs. 3e and S4b). Cells in the deep layer showed dendritic morphology (Fig. 3f) and expressed PHEX (Figs. 3g and S4b). These sequential changes in cellular morphology and the expression of stage-specific markers indicated that the current 3D culture system recapitulates the transition of osteoblasts to osteocytes.

Figure 3
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. (bg) 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.

Osteogenic induction of hiPSCs produced both stem cell-like cells and terminally differentiated cells

Aggregation and invasion into the gel were observed focally, and other cells remained on the gel with a sheet-like structure, suggesting that cells were heterogenous in terms of the differentiation status even at day 14. To characterize these heterogenous cells, their expression profiles were analyzed at the single cell level. The whole cell fraction (WC) and invading cell fraction (IC) were prepared as described in the Method section and subjected to scRNA sequencing. Data obtained from WC and IC were combined and analyzed using Seurat v4, which classified the induced cells into 15 clusters, as demonstrated by UMAP (Fig. 4a) and differentially expressed genes (DEGs) in each cluster were identified as (Fig. S5). Based on the contribution of WC- and IC-derived cells in each cluster (Fig. 4b), we were able to speculate the localization of cells in some clusters. For example, clusters 2, 4 and 6 were almost exclusively composed of WC-derived cells, indicating that cells in these clusters were on the surface of the gel. On the other hand, most cells in cluster 1 were derived from IC, indicating that these cells were inside the gel. Other clusters were composed of both populations but at different ratios.

Figure 4
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.

A pseudotime trajectory analysis was performed using Monocle 3 R to investigate the differentiation process and indicated cells in clusters 8, 10 and 13 were at the root of the trajectory and precursors of other cells (Fig. 4a, c). Among the differentially expressed genes (DEG) in these clusters, we identified several markers of SSCs, such as PRRX1 and PDGFRA genes19,20 (Fig. 4d, e). Cells in these clusters also expressed periostin (POSTN) and cathepsin K (CTSK) genes (Fig. 4d), which are markers for periosteal SSCs20,21. These data suggested that cells in clusters 8, 10 and 13 have progenitor potential. We also found that cells in these clusters expressed several early osteoblastic-lineage marker genes regulated by the TGFβ signal, such as cadherin 11 (CDH11), MMP2, and TGFBI22,23,24(Fig. 4d).

The pseudotime trajectory analysis also indicated that cells in cluster 5 were located between SSC-like cells and differentiated cells. Transglin (TAGLN), which regulates osteoblastic differentiation from precursors through actin reorganization25, was identified as a DEG in this cluster (Fig. 4d,e). CLIC3 gene was another DEG in this cluster (Fig. 4d,e) and recently identified as a lineage-specific gene regulating the differentiation of osteoblasts from MSCs26.

Cells in clusters 1 and 4 shared the expression of several osteoblast-related genes, although those in cluster 4 were on the surface of the gel and those in cluster 1 were inside. Some genes, such as TRIB1, CAVIN1, amphiregulin (AREG), and DEC1, were preferentially expressed in the surface cluster27,28,29,30, whereas KLF10 and TENT5A were equally expressed in both populations31,32. Mutations of TENT5A gene were recently identified in a hereditary bone disease, osteogenesis imperfecta33. Finally, although the number was limited, some cells in cluster 1 expressed the osteocyte marker gene sclerostin (SOST) and MMP1433,34.

These results indicated that the current induction method on type I collagen gel induced SSCs from hiPSCs, which produced differentiated cells on the surface and inside the gel.

Cell invasion required MMP activity

The results of the scRNA sequencing analysis suggested that the TGFβ signal plays a role in the maintenance of osteoblastic phenotype by regulating the expression of several genes, including MMP2. Vertical sections of induced cells with collagen gel at day 14 showed that phosphorylated-Smad3 was stained in cells on or just beneath the surface (Fig. 5a,b). The expression of MMP2 was also detected in these cells, but not in cells located in the deeper area of the gel (Fig. 5c,d). On the other hand, cells in the deeper area expressed MMP14 (Fig. 5e,f), which agrees with the scRNA sequencing data.

Figure 5
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 (gj) 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.

Next, a pan inhibitor of MMP, GM6001, was used to investigate the role of MMPs for cell invasion. Horizontal sections at day 14 showed the focal accumulation of cells in the gel (Fig. 5g,h), but this accumulation was almost completely inhibited by treatment with GM6001 (Fig. 5i,j). The same experiment was performed using MLO-Y4 cells, and the number of cells in the gel was clearly decreased by treatment with GM6001 (Fig. 5k,l).

TGFβ signal regulated the morphology and motility of hiPSC-derived osteogenic cells

Finally, we evaluated the effect of the TGFβ signal on cells isolated from 3D culture at day 14. Cells in WC showed heterogenous cell morphology, including osteoblast-like and osteocyte-like cells (Fig. 6a), and osteoblast-like cells vigorously proliferated again and produced a sheet-like structure, among which osteocytic cells rapidly migrated but rarely divided (Mov. S3). Cuboidal osteoblastic cells became dominant after treatment with TGFβ1 (Fig. 6b), whereas treatment with a TGFβ signal inhibitor (SB431542) increased cells with dendritic, osteocytic morphology (Fig. 6c). Immunostaining of COL I showed reduced COL 1 production upon inhibition of the TGFβ signal (Fig. 6d–i). Time-lapse imaging showed different cell behaviors. In control medium, cuboidal cells vigorously proliferated, making a sheet-like structure, and some dendritic cells became cuboidal (Fig. 6j and Mov. S4). On the other hand, SB431542 treatment reduced the proliferation of the cuboidal cells and enhanced the movement of dendritic cells (Fig. 6k and Mov. S5).

Figure 6
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) (ac) or antibody for COL I (df). (gi) 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.

These effects of the TGFβ signal on cellular behavior were also observed in experiments using MLO-Y4 cells. MLO-Y4 is an osteocytic cell line, but the morphology under standard culture condition is heterogenous, showing both polygonal osteoblastic and dendritic osteocytic morphologies (Fig. S6a). Under culture with TGFβ1, cells with polygonal morphology became dominant (Fig. S6b), whereas cells with dendritic morphology became dominant under culture with SB431542 (Fig. S6c).

These data indicated that the TGFβ signal maintains the osteoblastic phenotype of osteogenic-induced hiPSCs, and therefore reduction of the signal is required for the transition process from osteoblasts to osteocytes.



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