J Korean Soc Spine Surg.  2004 Jun;11(2):67-76. 10.4184/jkss.2004.11.2.67.

Mesenchymanl Stem Cell Based Intradiscal Gene Therapy: Therapeutic Implication in Degenerative Disc Disease

• Kim H
• Kwon UH
• Lee KI
• Song KH
• Shin SY
• Park SY
• Park JO
• Lee HM
• Moon SH

• KMID: 2097812
• DOI: http://doi.org/10.4184/jkss.2004.11.2.67

Abstract

STUDY DESIGN: In-vitro experiments using human mesenchymal stem cells (MSCs), intervertebral disc (IVD) cells and type 5 adenovirus/transforming growth factor-beta1 construct (Ad/TGF-beta1).
OBJECTIVES
To determine the effect of MSC-based gene therapy for matrix regeneration of IVD cells. SUMMARY OF LITERATURE REVIEW: MSCs are known to be multipotent in tissue regeneration. In degeneration of IVD, cellular replacement with genetic modification other than that of IVD cells may prove an enhanced mechanism for the regeneration of MATERIALS AND METHODS: MSCs and IVD cells were cultured and an adenovirus construct containing TGF-beta1 cDNA (Ad/TGF-beta1) was also produced. In the first step, the MSCs were transduced with Ad/TGF-beta1, then mixed with IVD cells in various proportions and three dimensionally cultured. [methyl-(3)H]Thymidine and [(35)S]Sulfur incorporation for DNA and proteoglycan synthesis, respectively, were measured. RT-PCR was performed to assess the aggrecan and collagen types I and II mRNA
RESULTS
Mixed cultures of MSC and IVD cells showed relatively similar amounts of newly synthesized proteoglycan compared with cultures of IVD cells only. In mixed cultures transduced with Ad/TGF-beta1, there were significant decreases in newly synthesized proteoglycan with increasing the proportions of MSCs, which was also found with the aggrecan and collagen type II mRNA expressions. However, the collagen type I mRNA expression increased with increased proportions of MSCs transduced with Ad/TGF-beta1.
CONCLUSION
Cell therapy with MSCs and IVD cells provided a mechanism for cellular augmentation. However, MSC-based gene therapy coupled with IVD cells did not maintain a chondrogenic phenotype.

Keyword

Mesenchymal stem cell; disc cell; TGF-b1; adenovirus; gene therapy

MeSH Terms

Adenoviridae
Aggrecans
Cell- and Tissue-Based Therapy
Collagen
Collagen Type I
Collagen Type II
DNA
DNA, Complementary
Genetic Therapy*
Humans
Intervertebral Disc
Mesenchymal Stromal Cells
Phenotype
Proteoglycans
Regeneration
RNA, Messenger
Stem Cells*
Transforming Growth Factor beta1
Aggrecans
Collagen
Collagen Type I
Collagen Type II
DNA
DNA, Complementary
Proteoglycans
RNA, Messenger
Transforming Growth Factor beta1

Figure

• 

  Fig. 1. (A) total DNA content in three dimensional culture of IVD cells and MSCs, (B)Newly synthesized proteoglycan in three dimensional culture of IVD cells and MSCs presented as percent control. Mixed cultures with IVD cells and MSCs equalled or quite decreased in newly synthesized proteoglycan comparing only IVD cell cultures (p 〈0.05).

• 

  Fig. 2. (A) total DNA content in three dimensional culture of IVD cells and Ad/TGF-β1-transduced MSCs, (B) Newly synthesized proteoglycan in three dimensional culture of IVD cells and Ad/TGF-β1-transduced MSCs presented as percent control (p 〈0. 05).

• 

  Fig. 3. RT-PCR for aggrecan, collagen type I, and collagen type II in three dimensional culture of IVD cells and MSCs. β-actin was used for normalization. Mixed ratio of IVD cells and MSCs are a) 1:0, b) 0:1, c)4:1, d) 1:1, e) 1:4.

• 

  Fig. 4. RT-PCR for aggrecan, collagen type I, and collagen type II in three dimensional culture of IVD cells and Ad-transduced MSCs. β-actin was used for normalization. a) only IVD cells, b) only MSC, c) only Ad/luc-transduced MSC [Mock], d) only Ad/TGF-β1-transduced MSC [Ad/TGF-β1-MSC], e) [IVD cells : Ad/TGF-β1-MSC] 1:1, f) [IVD cells : Ad/TGF-β1-MSC] 1:4.

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