By Feodor Price

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The effect of loading conditions on the gene
expression of mature osteoblasts and chondrocytes.

• By Feodor Price

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Outline

• Background
• - Embryonic Stem cells
• - Osteoblast/Chondrocyte differentiation
• - Compression Chamber
• Goals of the Research
• Protocol
• Results
• Summary

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Obtaining Embryonic Stem cells
What are ES cells?
Stem cell lecture by Paul Berg at Stanford
University
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ES cells can differentiate into many different
cell types
Spontaneous differentiation occurs when no growth
factors are added.
Future Introduction of Human ES cells
Stem cell lecture by Paul Berg at Stanford
University
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Proof of the feasibility of the differentiation
processes

• Growth factors
• Osteoblasts
• Vitamin D3 (VD3), ß-Glycerophosphate (ßGP),
  Ascorbic Acid (AA).
• Chondrocytes
• Transforming Growth factor ß1 (TGF-ß1), Bone
  morphogenic protein 2 (BMP-2), Insulin (INS), and
  Ascorbic Acid (AA)
• Specific markers for osteoblasts and
  chondrocytes.

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Differentiation Protocol in vitro
Chondrocyte Growth Factors added
Osteoblast Growth Factors added
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Compression Chamber

• Chamber in which hydrostatic pressure is created.
  

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Outline

• Background
• - Embryonic Stem cells
• - Osteoblast/Chondrocyte differentiation
• - Compression Chamber
• Goals of the Research
• Protocol
• Results
• Summary

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Goals of the Research

• Attempt to recapitulate in vivo environment
• Expose mature osteoblasts and chondrocytes to
  loading conditions.
• Examine the gene expression of 12 osteoblast or
  chondrocyte specific genes.
• Pilot project for future experiments using
  loading conditions.

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Outline

• Background
• - Embryonic Stem cells
• - Osteoblast/Chondrocyte differentiation
• - Compression Chamber
• Goals of the Research
• Protocol
• Results
• Summary

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Mature osteoblasts (top) And chondrocytes (bottom)
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Cells before loading
Kent and Nicole with chamber in backgroud
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Remove oilgt Lyse cellgt collect RNAgt
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Quantify RNAgt cDNA synthesisgt QPCR
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What is QPCR?

• PCR -gt amplify your DNA
• Real time
• Quantitative
• Standardized against GAPDH
• Infer back to gene expression.
• - cDNAgt mRNAgt Gene activity or expression

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Outline

• Background
• - Embryonic Stem cells
• - Osteoblast/Chondrocyte differentiation
• - Compression Chamber
• Goals of the Research
• Protocol
• Results
• Summary

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Results
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Outline

• Background
• - Embryonic Stem cells
• - Osteoblast/Chondrocyte differentiation
• - Compression Chamber
• Goals of the Research
• Protocol
• Results
• Summary

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Summary

• Protocols for osteogenesis and chondrogenesis
  were carried out successfully.
• RNA isolation, cDNA synthesis, and QPCR were all
  successful.
• Loading has effects on the gene expression of all
  three cell types.

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Future work

• Analysis of the effect of loading on the
  differentiation process itself.
• Microarray analysis of the results obtained so
  far.
• Further analysis of the QPCR results obtained so
  far.

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Acknowledgments

• Nicole zur Nieden
• Kent Paulson
• The Rancourt Lab
• The Hart Lab
• Markin-Flanagan Scholarship

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References

• zur Nieden, N.I., Kempka, G. and Ahr, H.J. (2003)
  In Vitro differentiation of embryonic stem cells
  into mineralized osteoblasts. Differentiation
  7118-27.
• Kramer, J. et al (2000) Embryonic stem
  cell-derived chondrogenic differentiation in
  vitro activation by BMP-2 and BMP-4. Mechanisms
  of Development 92193-205