3D-cultured human placenta-derived mesenchymal stem cell spheroids enhance ovary function by inducing folliculogenesis

Abstract

Placenta-derived mesenchymal stem cells (PD-MSCs) have numerous advantages over other adult MSCs that make them an attractive cell source for regenerative medicine. Here, we demonstrate the therapeutic effect of PD-MSCs in ovariectomized (Ovx) rats and compare their efficacy when generated via a conventional monolayer culture system (2D, naïve) and a spheroid culture system (3D, spheroid). PD-MSC transplantation significantly increased the estradiol level in Ovx rats compared with the non-transplantation (NTx) group. In particular, the estradiol level in the Spheroid group was significantly higher than that in the Naïve group at 2 weeks. Spheroid PD-MSCs exhibited a significantly higher efficiency of engraftment onto ovarian tissues at 2 weeks. The mRNA and protein expression levels of Nanos3, Nobox, and Lhx8 were also significantly increased in the Spheroid group compared with those in the NTx group at 1 and 2 weeks. These results suggest that PD-MSC transplantation can restore ovarian function in Ovx rats by increasing estrogen production and enhancing folliculogenesis-related gene expression levels and further indicate that spheroid-cultured PD-MSCs have enhanced therapeutic potential via increased engraftment efficiency. These findings improve our understanding of stem-cell-based therapies for reproductive systems and may suggest new avenues for developing efficient therapies using 3D cultivation systems.

Figure 1

Schematic illustrations of the 3D spheroid culture of PD-MSCs and the models used to test their effect on ovary function. PD-MSCs isolated from the chorionic plates of human placenta were seeded onto PDMS-based concave microwell arrays for generation of 3D spheroids. For transplantation into ovariectomized rats, spheroidal PD-MSCs and naïve PD-MSCs were directly injected into the remaining ovary. The therapeutic effects of PD-MSCs were assessed by analysis of E₂ production and the expression levels of folliculogenesis-related genes.

Figure 2

Morphologies and viabilities of PD-MSC spheroids. (a) Optical images of PD-MSC spheroids forming in concave microwells over time. Scale bars: 200 µm. (b) SEM images of spheroids on days 1 (left) and 3 (right) with magnified surface images showing differences in the junctions between neighboring cells (arrowheads). Scale bars: 10 nm. (c) Analysis of the diameter of PD-MSC spheroids over time. (d) Cell viabilities in spheroids cultured for 1 and 3 days. Scale bars: 200 µm. The quantification data (right) are expressed as the mean ± SE (n = 25).

Figure 3

Therapeutic effect of PD-MSCs in ovariectomized (Ovx) rats. (a) The graph represents the ratio of ovary weight to body weight in Ovx rats transplanted with naïve or spheroid PD-MSCs. (b) E₂ levels in plasma samples from transplanted and non-transplanted rats. (c) Quantification of the expression of human-specific Alu sequences in rat ovary tissues engrafted with naïve PD-MSCs or spheroidal PD-MSCs, as assessed by qRT-PCR. (d) Histological analysis of ovary tissues obtained from Ovx rats transplanted with or without naïve or spheroidal PD-MSCs (left). Quantification of follicles in rat ovary tissues according to PD-MSC transplantation (right). The arrows indicate follicles below 100 µm in size. Scale bar: 20 nm. The data are expressed as the mean ± SE. Symbols: ^*represent a significant difference compared to the NTx group (p < 0.05); ^#represents a significant difference between the Naïve and Spheroid groups (p < 0.05). Abbreviations: Ovx, ovariectomized (unilateral); Con, sham-surgery group; NTx, non-transplanted Ovx model; Naïve, naïve PD-MSC-transplanted group; and Spheroid, spheroid PD-MSC-transplanted group.

Figure 4

Analysis of oogenesis-related gene expression in ovary tissues of Ovx rats with or without PD-MSC transplantation. (a) The mRNA expression levels of Nanos3 (left), Nobox (middle) and Lhx8 (right) were assessed with qRT-PCR. (b) The protein expression levels of Nanos3, Nobox and Lhx8 were assessed with Western blot analysis. (c) Localization of Nanos3 and Lhx8 in rat ovary tissues, as assessed by double immunofluorescence (red  LNanos3; green = Lhx8; blue = nuclei, DAPI). Scale bar: 20 nm (x200 original magnification). (d) Localization of Nobox and stem121 in rat ovary tissues, as assessed with double immunofluorescence (red = Nobox; green  gstem121; blue tenuclei, DAPI). Scale bar: 20 µm (x100 original magnification). The data are expressed as the mean ± SE. Symbols: ^*represents a significant difference compared to the NTx group (p < 0.05); and ^#represents a significant difference between the Naïve and Spheroid groups (p < 0.05). Abbreviations: Ovx, ovariectomized (unilateral); Con, sham-surgery group; NTx, non-transplanted Ovx model; Naïve, naïve PD-MSC-transplanted group; and Spheroid, spheroid PD-MSC-transplanted group.