Journal List > J Korean Soc Spine Surg > v.11(2) > 1035642

Kim, Kwon, Kwang-Il-Lee, Song, Shin, Park, Park, Lee, and Moon: Mesenchymanl Stem Cell Based Intradiscal Gene Therapy: Therapeutic Implication in Degenerative Disc Disease

Abstract

Study Design

In- vitro experiments using human mesenchymal stem cells (MSCs), intervertebral disc (IVD) cells and type 5 adenovirus/transforming growth factor- β1 construct (A d/TGF- β1).

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 IVD cells.

Materials and Methods

MSCs and IVD cells were cultured and an adenovirus construct containing TGF- β1 cDNA (A d/TGF- β 1) was also produced. In the first step, the MSCs were transduced with A d/TGF- β1, 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 expressions

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 A d/TGF- β1, 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 A d/TGF- β1.

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.

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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).
jkss-11-67f1.tif
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).
jkss-11-67f2.tif
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.
jkss-11-67f3.tif
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.
jkss-11-67f4.tif
Table 1.
Primer sequence and reaction temperature
  primer sequence (5’ ->3’) annealing temperature (° C)
β-actin GGC GGA CTA TGA CTT AGT TG 53
  AAA CAA CAA TGT GCA ATC AA  
aggrecan GGA TCT AGC AGT GAG ACG TC 47
  CTG CAG CAG TTG ATT CTG AT  
collagen type I CCT GTC TGC TTC CTG TTA AC 48
  AGA GAT GAA TGC AAA GGA AA  
collagen type II CAG GAC CAA AGG GAC AGA AA 54
  TTG GTC CTT GCA TTA CTC CC  
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