Yonsei Med J.  2013 Jan;54(1):246-252. 10.3349/ymj.2013.54.1.246.

The Genetically Modified Polysialylated Form of Neural Cell Adhesion Molecule-Positive Cells for Potential Treatment of X-Linked Adrenoleukodystrophy

• Jang J
• Kim HS
• Kang JW
• Kang HC

• KMID: 1776946
• DOI: http://doi.org/10.3349/ymj.2013.54.1.246

Abstract

PURPOSE
Cell transplantation of myelin-producing exogenous cells is being extensively explored as a means of remyelinating axons in X-linked adrenoleukodystrophy. We determined whether 3,3',5-Triiodo-L-thyronine (T3) overexpresses the ABCD2 gene in the polysialylated (PSA) form of neural cell adhesion molecule (NCAM)-positive cells and promotes cell proliferation and favors oligodendrocyte lineage differentiation.
MATERIALS AND METHODS
PSA-NCAM+ cells from newborn Sprague-Dawley rats were grown for five days on uncoated dishes in defined medium with or without supplementation of basic fibroblast growth factor (bFGF) and/or T3. Then, PSA-NCAM+ spheres were prepared in single cells and transferred to polyornithine/fibronectin-coated glass coverslips for five days to determine the fate of the cells according to the supplementation of these molecules. T3 responsiveness of ABCD2 was analyzed using real-time quantitative polymerase chain reaction, the growth and fate of cells were determined using 5-bromo-2-deoxyuridine incorporation and immunocytochemistry, respectively.
RESULTS
Results demonstrated that T3 induces overexpression of the ABCD2 gene in PSA-NCAM+ cells, and can enhance PSA-NCAM+ cell growth in the presence of bFGF, favoring an oligodendrocyte fate.
CONCLUSION
These results may provide new insights into investigation of PSA-NCAM+ cells for therapeutic application to X-linked adrenoleukodystrophy.

Keyword

X-linked adrenoleukodystrophy; polysialylated form of the neural cell adhesion molecule cells; 3,3',5-Triiodo-L-thyronine; ABCD2 gene

MeSH Terms

ATP-Binding Cassette Transporters/*metabolism
Adrenoleukodystrophy/genetics/*therapy
Animals
Animals, Newborn
Bromodeoxyuridine
Cell Differentiation
Fibroblast Growth Factor 2/pharmacology
Fibronectins/metabolism
Immunohistochemistry
Neural Cell Adhesion Molecules/*genetics
Rats
Rats, Sprague-Dawley
Real-Time Polymerase Chain Reaction
Sialic Acids/metabolism
Stem Cells
Thyroid Hormones/*metabolism
Triiodothyronine/pharmacology
Fibronectins
Neural Cell Adhesion Molecules
Sialic Acids
Thyroid Hormones
Triiodothyronine
Fibroblast Growth Factor 2
Bromodeoxyuridine

Figure

• 

  Fig. 1 (A) From neonatal Sprague Dawley rat brain, abundant PSA-NCAM+ cells were isolated by Percoll density gradient method. (B) The culture and differentiation protocol for PSA-NCAM+ cells. (C) Progenitor cells identified using the nestin and PSA-NCAM markers. Scale bar=50 µm. PSA, polysialylated; NCAM, neural cell adhesion molecule; DAPI, 4',6-diamidine-2'-phenylindole dihydrochloride; T3, 3,3',5-Triiodo-L-thyronine; bFGF, basic fibroblast growth factor.

• 

  Fig. 2 In the PSA-NCAM+ cells, PSA disappeared in 7 days. Scale bar 50 µm. DIV, days in vitro. PSA, polysialylated; NCAM, neural cell adhesion molecule; DAPI, 4',6-diamidine-2'-phenylindole dihydrochloride.

• 

  Fig. 3 BrdU labeling shows that survival in cultures treated with both FGF2 and T3 was 9.5-fold higher than in the untreated controls (p=0.0075), T3 alone (p=0.0062) and 1.5-fold higher than cells treated with FGF2 alone (p=0.0433). *p<0.05; †p<0.01. BrdU, 5-bromo-2-deoxyuridine; T3, 3,3',5-Triiodo-L-thyronine; bFGF, basic fibroblast growth factor.

• 

  Fig. 4 (A) Oligodendrocytes identified using the O4 marker. (B) In PSA-NCAM+ cell, spheres grown with FGF2 and T3 differentiated into astrocytes (51%) and oligodendrocytes (44%), while only 5% neurons were observed. In the absence of T3 and FGF2 or with T3 only, only 20% oligodendrocytes were observed (p=0.0023), whereas 31% oligodendrocytes were observed in cultures treated with FGF2 only (p=0.035). Scale bar=50 µm. *p<0.05; †p<0.01. T3, 3,3',5-Triiodo-L-thyronine; bFGF, basic fibroblast growth factor; PSA, polysialylated; NCAM, neural cell adhesion molecule.

• 

  Fig. 5 The value of real-time quantitative polymerase chain reaction at the 40 ng/mL T3 and 10 ng/mL FGF2 dose. The induction of the ABCD2 gene in the PSA-NCAM+ cells was, 1.8-fold higher than in untreated controls (p=0.0325) and 1.7-fold higher than cells treated with FGF2 alone (p=0.0345). *p<0.05. T3, 3,3',5-Triiodo-L-thyronine; bFGF, basic fibroblast growth factor; PSA, polysialylated; NCAM, neural cell adhesion molecule.

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