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Differentiation of human mesenchymal stem cell spheroids under microgravity conditions.

Cerwinka WH, Sharp SM, Boyan BD, Zhau HE, Chung LW, Yates C - Cell Regen (Lond) (2012)

Bottom Line: 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.The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.

View Article: PubMed Central - PubMed

Affiliation: Children's Healthcare of Atlanta, Emory University School of Medicine, 5445Meridian Mark Road, Suite 420, Atlanta, GA 30342 USA ; Georgia Pediatric Urology, 5445 Meridian Mark Rd, Suite 420, Atlanta, GA 30342 USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Fig5: 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.

Mentions: Since mineralization appeared to occur on day 10, we further sought to determine if this mineralization was associated with additional markers of osteogenesis, alkaline phosphatase (ALP), nuclear vitamin D receptor (VDR) and membrane vitamin D receptor (ERp60) over the 10 day culture period. ALP staining was completely absent in 3 day spheroids, however, we did detect expression at 10 days under control and osteogenic conditions (Figure 5A). We also observed significant levels of nuclear VDR and ERp60 expression in the 3 day and 10 day control and osteogenic spheroids (Figure 5A). Robust Ki-67 expression was observed in undifferentiated and differentiated cultured conditions. (Figure 5B). Thus, it appears that these studies provide proof-of-principle that hMSC can be induced toward the osteogenic lineage under MG conditions.Figure 5


Differentiation of human mesenchymal stem cell spheroids under microgravity conditions.

Cerwinka WH, Sharp SM, Boyan BD, Zhau HE, Chung LW, Yates C - Cell Regen (Lond) (2012)

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.
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Related In: Results  -  Collection

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Fig5: 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.
Mentions: Since mineralization appeared to occur on day 10, we further sought to determine if this mineralization was associated with additional markers of osteogenesis, alkaline phosphatase (ALP), nuclear vitamin D receptor (VDR) and membrane vitamin D receptor (ERp60) over the 10 day culture period. ALP staining was completely absent in 3 day spheroids, however, we did detect expression at 10 days under control and osteogenic conditions (Figure 5A). We also observed significant levels of nuclear VDR and ERp60 expression in the 3 day and 10 day control and osteogenic spheroids (Figure 5A). Robust Ki-67 expression was observed in undifferentiated and differentiated cultured conditions. (Figure 5B). Thus, it appears that these studies provide proof-of-principle that hMSC can be induced toward the osteogenic lineage under MG conditions.Figure 5

Bottom Line: 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.The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.

View Article: PubMed Central - PubMed

Affiliation: Children's Healthcare of Atlanta, Emory University School of Medicine, 5445Meridian Mark Road, Suite 420, Atlanta, GA 30342 USA ; Georgia Pediatric Urology, 5445 Meridian Mark Rd, Suite 420, Atlanta, GA 30342 USA.

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.

No MeSH data available.


Related in: MedlinePlus