Differentiation of human mesenchymal stem cell spheroids under microgravity conditions

Abstract

To develop and characterize a novel cell culture method for the generation of undifferentiated and differentiated human mesenchymal stem cell 3D structures, we utilized the RWV system with a gelatin-based scaffold. 3 × 10(6) cells generated homogeneous spheroids and maximum spheroid loading was accomplished after 3 days of culture. Spheroids cultured in undifferentiated spheroids of 3 and 10 days retained expression of CD44, without expression of differentiation markers. Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively. Further characterization of osteogenic lineage, showed that 10 day spheroids exhibited stronger calcification than any other experimental group corresponding with significant expression of vitamin D receptor, alkaline phosphatase, and ERp60 . In conclusion this study describes a novel RWV culture method that allowed efficacious engineering of undifferentiated human mesenchymal stem cell spheroids and rapid osteogenic differentiation. The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.

Keywords: Differentiation; Mesenchymal stem cell; Osteogenesis; RWV culture; Tissue engineering.

Figure 1

Schematic of generation of multicellular spheroids . 2D cultured hMSC were suspended in growth media in the presence of scaffold. Cell suspensions containing scaffolds were transferred to RWV rotary cell culture system and cultured at 4 rounds per minute for designated time intervals. After designated time intervals multicellular spheroids were sectioned and examined for cellularity and differentiation status.

Figure 2

Quantification of hMSC loading onto gelatin scaffolds under MG conditions. (A) Number of cells loaded onto spheroids was determined by quantification of DNA content per spheroid over an 8 day period. (B) Number of cells that remained in suspension over an 8 day period. All data presented are the mean of three independent experiments ± SE. (C) H&E staining of day 3 spheroid at 10x and 40x magnification shows multi-cellularity and absence of necrosis. (D) TEM images of day 3 spheroid show cell structure was maintained in MG. Images shown are representative of 4 individual experiments.

Figure 3

Differentiation of hMSC spheroids cultured under MG conditions . (A) Immunohistochemistry analysis of spheroids for stem cells makers CD44, CD133, CD166, and differentiation markers von Kossa, oil red O, collagen-II showed that day 3 spheroids retained CD44, CD133, and CD166 expression and lacked differentiation markers. (B) Day 10 spheroids retain CD44 expression and were positive for oil red O staining. Images shown are representative of 4 individual experiments.

Figure 4

Induced differentiation of hMSC spheroids cultured under MG conditions. (A) Immunohistochemistry analysis of hMSC cultured for 10 days in A, chrondrogenic media shows negative staining for collagen. (B) hMSC spheroids cultured under adipogenic conditions show positive oil read O staining. (C) hMSC spheroids cultured under osteogenic conditions were positive with von Kossa staining. Images shown are representative of 4 individual experiments.

Figure 5

Osteogenic differentiation of hMSC spheroids cultured under MG conditions. (A) Immunohistochemical analysis of alkaline phosphatase (AP), vitamin D receptor (VDR), and ERp60 expression in hMSC spheroids after day 3 and day 10 with and without osteogenic conditions. (B) Immunohistochemical analysis of proliferation marker Ki-67 at day 3 and day 10. Images shown are representative of 4 individual experiments.