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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

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.
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Fig3: 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.

Mentions: Numerous reports have provided compelling evidence that hMSC enhance organ repair. However, a major obstacle is the delivery of a sufficient number of undifferentiated cells to the site of injury. Therefore, it is essential that MG conditions do not alter hMSC multi-lineage differentiation potential. To determine whether 3 day spheroids, cultured under maintenance conditions alone, retained stem cell markers, we evaluated expression of stem cell markers (CD44, CD133, CD166) and common markers for osteogenic, adipogenic, and chondrogenic differentiation by staining for von Kossa, oil red O, collagen II, and aggrecan, respectively over an additional 10 day period. Spheroids harvested after three days showed robust expression of CD44, CD133, and CD166 (Figure 3A), which is similar to hMSC cells cultured on 2D surfaces [16]; however, spheroids harvested after 10 days demonstrated a loss of CD133 and CD166 expression. Additionally, 10 day spheroids did not exhibit von Kossa staining or collagen II/aggrecan expression, however, we did observe low levels of oil red O staining (Figure 3B).Figure 3


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230401&req=5

Fig3: 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.
Mentions: Numerous reports have provided compelling evidence that hMSC enhance organ repair. However, a major obstacle is the delivery of a sufficient number of undifferentiated cells to the site of injury. Therefore, it is essential that MG conditions do not alter hMSC multi-lineage differentiation potential. To determine whether 3 day spheroids, cultured under maintenance conditions alone, retained stem cell markers, we evaluated expression of stem cell markers (CD44, CD133, CD166) and common markers for osteogenic, adipogenic, and chondrogenic differentiation by staining for von Kossa, oil red O, collagen II, and aggrecan, respectively over an additional 10 day period. Spheroids harvested after three days showed robust expression of CD44, CD133, and CD166 (Figure 3A), which is similar to hMSC cells cultured on 2D surfaces [16]; however, spheroids harvested after 10 days demonstrated a loss of CD133 and CD166 expression. Additionally, 10 day spheroids did not exhibit von Kossa staining or collagen II/aggrecan expression, however, we did observe low levels of oil red O staining (Figure 3B).Figure 3

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