Enrichment of committed human nucleus pulposus cells expressing chondroitin sulfate proteoglycans under alginate encapsulation.
OBJECTIVE
Intervertebral disc (IVD) degeneration is associated with a malfunction of the nucleus pulposus (NP). Alginate culturing provides a favorable microenvironment for the phenotypic maintenance of chondrocyte-like NP cells. However, NP cells are recently evidenced to present heterogeneous populations, including progenitors, fibroblastic cells and primitive NP cells. The aim of this study is to profile the phenotypic changes of distinct human NP cells populations and describe the dynamic expression of chondroitin sulfate glycosaminoglycans (CS-GAGs) in extended alginate encapsulation.


METHODS
Non-degenerated (ND-NPC) and degenerated (D-NPC) NP cells were expanded in monolayers, and subject to 28-day culture in alginate after serial passaging. CS-GAG compositional expression in monolayer-/alginate-cultured NP cells was evaluated by carbohydrate electrophoresis. Cellular phenotypic changes were assessed by immunologic detection and gene expression analysis.


RESULTS
Relative to D-NPC, ND-NPC displayed remarkably higher expression levels of chondroitin-4-sulfate GAGs over the 28-day culture. Compared with monolayer culture, ND-NPC showed increased NP marker expression of KRT18, KRT19, and CDH2, as well as chondrocyte markers SOX9 and MIA in alginate culture. In contrast, expression of fibroblastic marker COL1A1, COL3A1, and FN1 were reduced. Interestingly, ND-NPC showed a loss of Tie2+ but gain in KRT19+/CD24+ population during alginate culture. In contrast, D-NPC showed more consistent expression levels of NP surface markers during culture.


CONCLUSIONS
We demonstrate for the first time that extended alginate culture selectively enriches the committed NP cells and favors chondroitin-4-sulfate proteoglycan production. These findings suggest its validity as a model to investigate IVD cell function.
Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region.
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Y
Sun
Advanced Technology Research Institution of China Science Institution, Shenzhen, China.
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Lv
Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region.
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L
Zhou
Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region; Department of Biochemistry, The University of Hong Kong, Hong Kong Special Administrative Region.
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Tam
Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region; Department of Biochemistry, The University of Hong Kong, Hong Kong Special Administrative Region.
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F
Lv
Department of Biochemistry, The University of Hong Kong, Hong Kong Special Administrative Region.
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D
Chan
Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.
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H
Wang
Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China.
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Z
Zheng
Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region. Electronic address: cheungmc@hku.hk.
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Cheung
Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region. Electronic address: vicleung@hku.hk.
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VY
Leung
D000293:Adolescent; D000328:Adult; D000368:Aged; D000464:Alginates; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D020723:Glucuronic Acid; D006025:Glycosaminoglycans / Q000096:biosynthesis; D006603:Hexuronic Acids; D006801:Humans; D007403:Intervertebral Disc / Q000473:pathology*; D055959:Intervertebral Disc Degeneration / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D010641:Phenotype; D054457:Tissue Scaffolds
D000464:Alginates; D011508:Chondroitin Sulfate Proteoglycans; D006025:Glycosaminoglycans; D006603:Hexuronic Acids; D020723:Glucuronic Acid
Alginate; Chondroitin sulfate; Intervertebral disc degeneration; Nucleus pulposus; Proteoglycan