Effect of spheroid size on gene expression profiles of a mouse mesenchymal stem cell line in spheroid culture

1. Introduction

Bone regeneration constitutes a significant medical hurdle, as inadequate healing can lead to trauma, tumor development, and osteomyelitis. Mesenchymal stem/stromal cells (MSCs) are multipotent somatic stem cells with the potential to differentiate into various mesodermal cells, including osteoblasts and chondrocytes [1]. In addition, MSCs produce a number of trophic factors that can contribute to bone repair [2]. Therefore, MSCs have become an attractive cell source and are widely employed in the development of bone repair strategies [3–5].

Despite being a commonly used and traditional culture method, two-dimensional (2D) monolayer cell cultures do not offer a physiologically comparable environment for cell growth. In fact, the artificial culture conditions of cell monolayers can cause a reduction or loss of characteristics and traits in vitro, including self-renewal, replication, colony-forming efficiency, and differentiation capacity. Three-dimensional (3D) spheroid cultures, on the other hand, are better able to recapitulate the conditions found in vivo, and are thus better able to maintain these characteristics [6]. Further, MSCs cultured in spheroids have enhanced trophic activity compared to MSCs in monolayers. Therefore, MSC spheroids are a potential therapeutic source for bone regenerative medicine. However, while it is known that bone regeneration requires a complex interplay between molecules such as FGF, PDGF, TGF-β, VEGF, Wnt [7], factors that increase trophic factor expression in MSC spheroids are not fully understood.

A previous study that examined the link between cell density and alterations in MSC function in monolayer culture revealed elevated levels of proliferation-related genes at low cell density, and immunosuppression, migration, and cell motility-related genes at high cell density [8]. Thus, spheroid size may influence the expression of trophic factors. Although various spheroid sizes have been used in MSC spheroid research, the impact of spheroid size on trophic factor expression has not been fully investigated.

Here, we investigated the differential expression of genes in monolayer and spheroid cultures and the effect of cell number on trophic factor expression in the mouse MSC cell line, KUM10.

2. Materials and methods

2.2. RNA isolation and RNA-seq analysis

RNA-seq was used to screen the transcriptional profiles of monolayer and spheroid cells. To identify genes that were differentially expressed between monolayer and spheroid cells, total RNA extracted from 3 monolayer and 3 spheroid culture samples cultured at 5 × 10⁵ cells for 5 days was isolated using Trizol (Thermo Scientific) and a spin column (Direct-zol MicroPrep kit, Zymo Research, Orange, CA, USA). The amount of RNA was measured using a spectrophotometer (Denovix, DE, USA) and the quality was checked on the Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) using an RNA 6000 Nano Chip. RNA-seq was performed on the extracted RNA and RNA sequencing was conducted using DNBSEQ (DNBSEQ Technology platform) before sending the RNA samples to BGI Japan for RNA-seq analysis. Differentially expressed genes were defined by default as genes with an false discovery rate (FDR) ≤ 0.001 and fold change >2 in RNA-seq analysis [9,10].

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Table 1

Primer sequences

Gene		Sequence (5 ′ -3 ′ )	Product size (bp)
Bmp2	F	AAGGCACCCTTTGTATGTGG	211
	R	GCTAAGCTCAGTGGGGACAC
Fgf9	F	ACAACACCTACTCTTCCAACCT	114
	R	TCTGGTGCCGTTTAGTCCTG
Fgf18	F	TCTGGTTGGTATGTGGGCTTC	158
	R	CGGGATCGCTTGGTGACTG
Ngf	F	ACTTCCAGGCCCATGGTACA	82
	R	CCCATGTGGAAGACTGGGTG
Pdgfa	F	GGGAACTGAACAGGTGGGAG	88
	R	CCACGTAAGGCCATCAGGAA
Pdgfb	F	CCAAAGGCAAGCACCGAAAG	180
	R	CATCGAGACAGACGGACGAG
Tgfb1	F	CTCCCGTGGCTTCTAGTGC	133
	R	GCCTTAGTTTGGACAGGATCTG
Vegfa	F	CACTGGACCCTGGCTTTACT	79
	R	TCTGCTCTCCTTCTGTCGTG
Vegfc	F	AGAAGTATGCCGCTGTGTCC	196
	R	CCCGCAGTAATCCACATGGT
Wnt4	F	CATCGAGGAGTGCCAATACCA	73
	R	AAAGACAGGGAGGGAGTCCAG
Wnt5a	F	CAATGAAGCAGGCCGTAGGA	77
	R	TACAGGAGCCAGACACTCCA
Wnt10a	F	TGCAAGTGAGCAGACCCAAG	138
	R	GCTGTAAGAGCCAACCACCT
Gapdh	F	AACTTTGGCATTGTGGAAGG	223
	R	ACACATTGGGGGTAGGAACA

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Fig. 1.

RNA-seq analyses of DEGs in mesenchymal stem cell monolayers and spheroids. Venn diagram analysis of DEGs. The region of overlap among the three circles represents the common but DEGs among the three datasets. DEGs, differentially expressed genes.

3. Results

3.2. Effect of spheroid size on trophic factor expression

The cross-section area of spheroids increased with the number of seeded cells (Fig. 2A–C). Spheroids in the M and L groups had significantly larger areas than those in the S group (Fig. 2D). Further, spheroids in the L group had significantly larger areas than those in the S and M groups (Fig. 2D).

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Fig. 2.

Spheroid morphology and size. Morphology (A–C) and area (D) of spheroids. Scale bar = 500 μm.

The effect of spheroid size on trophic factor-related gene expressions is shown in Fig. 3. Although no significant differences were observed in Bmp2 levels (Fig. 3A), Fgf9 and Fgf18 were significantly elevated in the L group compared to the S and M groups (Fig. 3B, C), while Ngf expression was significantly increased in the S group compared to the M group (Fig. 3D). No significant differences were noted in Pdgfa levels (Fig. 3E). However, Pdfb was significantly elevated in the S and M groups compared to the L group (Fig. 3F). Meanwhile, Tgfb1 levels were significantly higher in the S group than the M and L groups, Vegfa and Vegfc was significantly increased in the L group compared to the S group (Fig. 3H, I), and Wnt4 was significantly elevated in the S and L groups than the M group (Fig. 3J). While no significant differences were noted in Wnt5a expression (Fig. 3K), Wnt10a levels were significantly increased in the L group compared to the S and M groups (Fig. 3L).

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Fig. 3.

Effect of spheroid size on trophic factor-related gene expression. qPCR analysis results for Bmp2 (A), Fgf9 (B), Fgf18 (C), Ngf (D), Pdgfa (E), Pdgfb (F), Tgfb1 (G), Vegfa (H), Vegfc (I), Wnt4 (J), Wnt5a (K), and Wnt10a (L).

RNA-seq analysis of KUM10 monolayer and spheroid cultures revealed significantly elevated expression of Bmp2, Pdgfb, Tgfb1, Vegf, and Wnt5a in spheroid compared to monolayer cells. Expression of Fgf9, Fgf18, Ngf, Pdgfa, Vegfc, Wnt4b, and Wnt10b was also increased in spheroid compare to monolayer cultures. Further, large spheroids showed higher expression of Fgf9, Fgf18, Vegfa, Vegfc, and Wnt10a than small spheroids. In contrast, small spheroids showed higher expression of Pdgfb and Tgfb1 than large spheroids. Thus, spheroid size is an important regulator of the expression of several trophic factors.

Previous studies have reported that multiple pathways contribute to bone regeneration via bone formation and angiogenesis. While members of the TGF-β superfamily play an important role in bone regeneration [11,12], VEGF/PDGF families are pivotal for mesenchymal proliferation, chemotaxis and angiogenesis during the bone repair process [13,14]. Wnt ligands, on the other hand, contribute to bone development and homeostasis via both β-catenin-dependent and -independent pathways [15–17]. For example, Wnt-β-catenin signaling plays a crucial role in committing precursors to the osteoblast lineage and facilitates the proliferation and differentiation of osteoblast precursors [18–21]. Previous studies have reported that BMP-2, TGF-β1, VEGF, Wnt5a and PDGF-BB levels increase in spheroid cultures [22–26]. Consistent with these reports, we observed elevated levels of Bmp2, Tgfb1, Pdgfb, and Vegfa in spheroid compared to monolayer cultures. In addition, RNA-seq analysis revealed increased levels of Fgf9, Fgf18, Ngf, Pdgfa, Wnt4, and Vegfc in spheroid cultures. FGF9 and FGF18 are known to induce the proliferation and differentiation of MSCs into osteoblasts [27,28]. Further, the combination of FGF9 and VEGF promotes both bone formation and angiogenesis in vivo [29]. Co-administration of FGF18 with BMP2 has been shown to strongly enhance bone formation in critical size bone defects [30]. Meanwhile, NGF stimulates osteoblast proliferation in vitro [31] and promotes the repair of rabbit mandible fracture in vivo [32]. Further, MSC-derived VEGFC promotes angiogenesis and skin wound healing [33], while VEGF-C facilitates osteogenic differentiation and mineralization of MSCs [34]. PDGF-AA and Wnt-4 promote osteogenic differentiation of MSCs [35,36], while Wnt10a inhibits adipogenesis and stimulates osteoblast genesis through β-catenin [37]. Thus, the trophic factors found to be increased in spheroid culture play a pivotal role in bone regeneration.

As there is no standardized spheroid size, various sized spheroids have been applied to bone regeneration studies in animal models [38–40]. In our study, we evaluated the impact of spheroid size on trophic factor gene expression and found that some trophic factor-related genes exhibit spheroid size-dependent profiles. Elevated levels of angiogenic factors such as Fgf9, Vegfa and Vegfc and the osteogenic factor Fgf18 were observed in large spheroids. In contrast, Pdgfb and Tgfb1, which play an important role in bone healing, especially in the early phase, were elevated in small spheroids. PDGF-BB strongly stimulates chemotaxis in MSCs [41,42], while TGF-β promotes mesenchymal condensation of osteogenic progenitor cells [43,44]. Our results suggest that size may affect the therapeutic potential of spheroids, indicating the need to standardize spheroid size to optimize therapeutic conditions.

A previous study reported that cell density in monolayer culture affects β-catenin signaling: β-catenin was located at the adherens junction at low cell density, but was translocated to the nucleus at high density in mouse embryonic stem cells [45]. β-catenin-translocated nuclei show increased FGF9, FGF18 and VEGF production [46–48]. Moreover, β-catenin suppress TGF-β signaling [49]. Therefore, alternation of β-catenin signaling may partly explain the spheroid size-dependent differential gene expression profile in our study. Further investigation of the signal pathway underlying this mechanism, including β-catenin signaling, is needed.

5. Conclusions

We found that several trophic factors were elevated in MSC spheroids using RNA-seq and that spheroid size may impact trophic factor expression. Our results will be useful for future studies assessing the utility of MSC spheroids for treating bone injury.