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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

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.
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Fig4: 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.

Mentions: Spheroids cultured in chondrogenic media demonstrated histological changes on H&E stain (data not shown) such as clear cell appearance and cell aggregates indicating imminent differentiation. While CD133 and CD166 expression was undetectable, CD44 expression was detected throughout (Figure 4A). Expression of chondrogenic marker collagen type II and aggrecan was also absent after 3 and 10 days of culture in both control and chondrogenic media (Additional file 1: Figure S1), however, there were significant levels of adipogenic marker oil red O staining (Figure 4A).Figure 4


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230401&req=5

Fig4: 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.
Mentions: Spheroids cultured in chondrogenic media demonstrated histological changes on H&E stain (data not shown) such as clear cell appearance and cell aggregates indicating imminent differentiation. While CD133 and CD166 expression was undetectable, CD44 expression was detected throughout (Figure 4A). Expression of chondrogenic marker collagen type II and aggrecan was also absent after 3 and 10 days of culture in both control and chondrogenic media (Additional file 1: Figure S1), however, there were significant levels of adipogenic marker oil red O staining (Figure 4A).Figure 4

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