Introduction: Intervertebral disc (IVD) degeneration is suggested to begin from the
nucleus pulposus (NP). Evidence from various studies highlights mesenchymal stem cells
(MSC), in most cases using bone marrow derived MSC, as a potential stem cell source
for NP regeneration. However MSC can be isolated from many sources with various
characteristics. There are indications that fetal or close to fetal tissue sources contain
MSC with relatively undifferentiated phenotype with respect to MSC from adult sources.
Moreover, umbilical cord (C)-MSC may have better chondrogenic differentiation potential
than bone marrow (B)-MSC. We hypothesize CMSC are different from BMSC, and more
efficient than BMSC in stimulating NP regeneration.
Methods: MSC were isolated from human bone marrow and umbilical cord with
corresponding ethical approval. BMSC and CMSC were characterized for cell surface
marker expression profile and differentiation potential.. RT-PCR of interest genes in NP
cells isolated from scoliosis and degenerate discs was performed to search for NP
degeneration indicators. Conditioned media (CM) was collected from confluent MSC
monolayer, and used for stimulation of four batches of degenerated NP cells isolated from
human degenerative intervertebral discs. Cell proliferation and cytotoxicity were assessed
by MTT assay. Proteoglycan content were measured by DMMB assay. Gene expression
of a series of degeneration related molecules including ACAN, SOX9, CDH2, CD55,
KRT19, KRT18, FBLN1 and MGP, and fibrosis related molecules, including MMP12,
HSP47, COL1A1, COL3A1 and FN1, of NP cells in MSC-CM were determined by real- time RT-PCR. All results were normalized to the control cells in basal medium. The
expression of discogenic, chondrogenic and osteogenic markers on BMSC and CMSC
were compared by RT-PCR.
Results and Conclusion: CMSC were similar to BMSC and fulfilled the minimum
criteria of MSC, however the expression of CD146, CD106 and Stro-1 was different, and
BMSC had a spontaneous osteogenesis tendency while CMSC expressed chondrogenic
marker even without TGF-beta stimulation. BMSC demonstrated a paracrine effect on
modulating human degenerated NP cells towards a non-degenerative phenotype in
stimulating cell proliferation, slightly enhancing proteoglycan production, upregulating
KRT19 while downregulating MMP12. Compared with BMSC, a higher paracrine effect of
CMSC was disclosed in modulating the phenotype of NP cells in all aspects tested, and
an intrinsic higher expression on CMSC of ‘potential NP markers’, including KRT19,
KRT18 and CD55, but lower expression of osteogenic markers, including RUNX2 and
ALPL, was revealed, which indicate a higher potential of CMSC for future clinical
application to treat IVD degeneration diseases. KRT19 and MMP12 were also confirmed
to be the highest differentially expressed candidate genes between cultured scoliosis and
degenerated human NP cells, indicating a high indicator potential of NP degeneration.
Furthermore, a subpopulation was detected in the degenerated NP cells that possessed
macrophage-like phenotype and activities, which may play a role in the pathogenesis of
IVD degeneration. In conclusion, studies in this thesis highlighted CMSC as a superior
source than BMSC for IVD repair. Further investigations into the active agents in the
conditioned media and the signalling pathway may help to elucidate the mechanism of
the effect. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/161570 |
| Date | January 2012 |
| Creators | Lü, Fengjuan., 吕凤娟. |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B48079753 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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