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Postgraduate Thesis: Potential of bone marrow and umbilical cord derived mesenchymal stem cells in intervertebral disc repair
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TitlePotential of bone marrow and umbilical cord derived mesenchymal stem cells in intervertebral disc repair
 
AuthorsLü, Fengjuan.
吕凤娟.
 
Issue Date2012
 
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
 
AbstractIntroduction: 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.
 
DegreeDoctor of Philosophy
 
SubjectStem cells - Therapeutic use.
Intervertebral disk prostheses.
 
Dept/ProgramOrthopaedics and Traumatology
 
DC FieldValue
dc.contributor.authorLü, Fengjuan.
 
dc.contributor.author吕凤娟.
 
dc.date.hkucongregation2012
 
dc.date.issued2012
 
dc.description.abstractIntroduction: 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.
 
dc.description.naturepublished_or_final_version
 
dc.description.thesisdisciplineOrthopaedics and Traumatology
 
dc.description.thesisleveldoctoral
 
dc.description.thesisnameDoctor of Philosophy
 
dc.identifier.hkulb4807975
 
dc.languageeng
 
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)
 
dc.relation.ispartofHKU Theses Online (HKUTO)
 
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.
 
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License
 
dc.source.urihttp://hub.hku.hk/bib/B48079753
 
dc.subject.lcshStem cells - Therapeutic use.
 
dc.subject.lcshIntervertebral disk prostheses.
 
dc.titlePotential of bone marrow and umbilical cord derived mesenchymal stem cells in intervertebral disc repair
 
dc.typePG_Thesis
 
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<item><contributor.author>L&#252;, Fengjuan.</contributor.author>
<contributor.author>&#21525;&#20964;&#23071;.</contributor.author>
<date.issued>2012</date.issued>
<description.abstract>&#65279;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 &#8216;potential NP markers&#8217;, 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.</description.abstract>
<language>eng</language>
<publisher>The University of Hong Kong (Pokfulam, Hong Kong)</publisher>
<relation.ispartof>HKU Theses Online (HKUTO)</relation.ispartof>
<rights>The author retains all proprietary rights, (such as patent rights) and the right to use in future works.</rights>
<rights>Creative Commons: Attribution 3.0 Hong Kong License</rights>
<source.uri>http://hub.hku.hk/bib/B48079753</source.uri>
<subject.lcsh>Stem cells - Therapeutic use.</subject.lcsh>
<subject.lcsh>Intervertebral disk prostheses.</subject.lcsh>
<title>Potential of bone marrow and umbilical cord derived mesenchymal stem cells in intervertebral disc repair</title>
<type>PG_Thesis</type>
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<description.thesisname>Doctor of Philosophy</description.thesisname>
<description.thesislevel>doctoral</description.thesislevel>
<description.thesisdiscipline>Orthopaedics and Traumatology</description.thesisdiscipline>
<description.nature>published_or_final_version</description.nature>
<date.hkucongregation>2012</date.hkucongregation>
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