The Experts below are selected from a list of 207 Experts worldwide ranked by ideXlab platform
Irving M. Shapiro - One of the best experts on this subject based on the ideXlab platform.
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Hypoxia activates MAPK activity in rat nucleus pulposus cells: regulation of Integrin expression and cell survival.
Spine, 2005Co-Authors: Makarand V. Risbud, Asha Guttapalli, Todd J. Albert, Irving M. ShapiroAbstract:OBJECTIVE The aim of the present study was to investigate whether activation of MAPK subtypes ERK and p38 influences Integrin expression and promotes nucleus pulposus cell survival in hypoxia. SUMMARY OF BACKGROUND DATA We have recently shown that in a low oxygen environment, rat nucleus pulposus cells activate phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways. However, the role of these signaling pathways in regulating cell matrix interactions is not known. METHODS Rat nucleus pulposus cells were cultured in hypoxia (2% O2) or normoxia (20% O2). Western blotting and kinase assay were used to analyze expression of MAPK signaling molecules. Cell attachment to collagen was studied using an adhesion assay, whereas flow cytometry and RT-PCR were performed to quantify Integrin receptor expression. RESULTS In a hypoxic environment (2% O2), rat nucleus pulposus cells showed a persistent phosphorylation of p38 and ERK proteins; pERK catalyzed the phosphorylation of Elk1-Gst fusion protein. When ERK activity was blocked, cell adhesion to Type II collagen, one of the major extracellular matrix proteins in the nucleus pulposus tissue, was impaired. A similar inhibitory effect on collagen adhesion was observed when nucleus pulposus cells were treated with an antibody to Alpha2 Integrin. Furthermore, when ERK activity was inhibited, there was a decrease in Alpha2 Integrin mRNA expression. In contrast to ERK, inhibition of p38 activity did not modulate Alpha2 Integrin subunit mRNA expression. Likewise, inhibition of ERK, but not p38, resulted in downregulation of nucleus pulposus Alpha2 Integrin protein levels and blocked cell survival in hypoxia. CONCLUSIONS Hypoxia activated MAPK signaling pathway activity in nucleus pulposus cells. ERK, but not p38, regulated Alpha2 Integrin expression and cell survival.
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differentiation of mesenchymal stem cells towards a nucleus pulposus like phenotype in vitro implications for cell based transplantation therapy
Spine, 2004Co-Authors: Makarand V. Risbud, Asha Guttapalli, Todd J. Albert, Edward J Vresilovic, Alan S Hillibrand, Alexander R Vaccaro, Irving M. ShapiroAbstract:Objective Because mesenchymal stem cells can differentiate into chondrocyte-like cells, we ask the question, can mesenchymal stem cells commit to the nucleus pulposus phenotype? Background Back pain, a significant source of morbidity in our society, is linked to degenerative changes of the intervertebral disc. Absence of suitable graft tissue limits therapeutic approaches for repair of disc tissue. For this reason, there is considerable interest in developing "tissue engineering" strategies for the regeneration of the nucleus pulposus. Methods Rat mesenchymal stem cells were immobilized in 3-dimensional alginate hydrogels and cultured in a medium containing transforming growth factor-beta1 under hypoxia (2% O2) and normoxia (20% O2). Mesenchymal stem cells were examined by confocal microscopy to evaluate their viability and metabolic status after labeling with Celltracker green, a thiol sensitive dye, and Mitotracker red, a dye sensitive to the mitochondrial membrane potential. Flow cytometry, semiquantitative reverse transcription polymerase chain reaction and Western blot analysis were carried out to evaluate phenotypic and biosynthetic activities and the signaling pathways involved in the differentiation process. Results Under hypoxic conditions, mesenchymal stem cells formed large aggregates and exhibited positive Celltracker and Mitotracker signals. Glucose transporter-3, matrix metalloproteinase-2, collagen type II and type XI, and aggrecan mRNA and protein expression was upregulated, whereas there was no change in the levels of decorin, biglycan, fibromodulin, and lumican. Hypoxia maintained the expression of CD44 (hyaluronan receptor), ALCAM (CD166), and endoglin (transforming growth factor-beta receptor). Likewise, expression of beta3 and Alpha2 Integrin was upregulated. Transforming growth factor-beta treatment increased MAPK activity and Sox-9, aggrecan, and collagen type II gene expression. Basal levels of the phosphorylated MAPK isoform ERK1/2, but not p38, were higher under hypoxic conditions than normoxia, and its activation was further augmented by treatment of cells with transforming growth factor-beta. In hypoxia, transforming growth factor-beta sustained phosphorylated p38 expression for an extended time period. Pharmacological inhibition of ERK1/2 and p38 enzymatic activity resulted in a decrease in Sox-9, aggrecan, and collagen type II mRNA levels. Conclusions Our results indicate that hypoxia and transforming growth factor-beta drive mesenchymal stem cell differentiation towards a phenotype consistent with that of the nucleus pulposus. Measurement of selected signaling molecules and response to specific inhibitors suggest involvement of MAPK signaling pathways. It is concluded that mesenchymal stem cells could be used to repopulate the damaged or degenerate intervertebral disc.
Makarand V. Risbud - One of the best experts on this subject based on the ideXlab platform.
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Hypoxia activates MAPK activity in rat nucleus pulposus cells: regulation of Integrin expression and cell survival.
Spine, 2005Co-Authors: Makarand V. Risbud, Asha Guttapalli, Todd J. Albert, Irving M. ShapiroAbstract:OBJECTIVE The aim of the present study was to investigate whether activation of MAPK subtypes ERK and p38 influences Integrin expression and promotes nucleus pulposus cell survival in hypoxia. SUMMARY OF BACKGROUND DATA We have recently shown that in a low oxygen environment, rat nucleus pulposus cells activate phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways. However, the role of these signaling pathways in regulating cell matrix interactions is not known. METHODS Rat nucleus pulposus cells were cultured in hypoxia (2% O2) or normoxia (20% O2). Western blotting and kinase assay were used to analyze expression of MAPK signaling molecules. Cell attachment to collagen was studied using an adhesion assay, whereas flow cytometry and RT-PCR were performed to quantify Integrin receptor expression. RESULTS In a hypoxic environment (2% O2), rat nucleus pulposus cells showed a persistent phosphorylation of p38 and ERK proteins; pERK catalyzed the phosphorylation of Elk1-Gst fusion protein. When ERK activity was blocked, cell adhesion to Type II collagen, one of the major extracellular matrix proteins in the nucleus pulposus tissue, was impaired. A similar inhibitory effect on collagen adhesion was observed when nucleus pulposus cells were treated with an antibody to Alpha2 Integrin. Furthermore, when ERK activity was inhibited, there was a decrease in Alpha2 Integrin mRNA expression. In contrast to ERK, inhibition of p38 activity did not modulate Alpha2 Integrin subunit mRNA expression. Likewise, inhibition of ERK, but not p38, resulted in downregulation of nucleus pulposus Alpha2 Integrin protein levels and blocked cell survival in hypoxia. CONCLUSIONS Hypoxia activated MAPK signaling pathway activity in nucleus pulposus cells. ERK, but not p38, regulated Alpha2 Integrin expression and cell survival.
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differentiation of mesenchymal stem cells towards a nucleus pulposus like phenotype in vitro implications for cell based transplantation therapy
Spine, 2004Co-Authors: Makarand V. Risbud, Asha Guttapalli, Todd J. Albert, Edward J Vresilovic, Alan S Hillibrand, Alexander R Vaccaro, Irving M. ShapiroAbstract:Objective Because mesenchymal stem cells can differentiate into chondrocyte-like cells, we ask the question, can mesenchymal stem cells commit to the nucleus pulposus phenotype? Background Back pain, a significant source of morbidity in our society, is linked to degenerative changes of the intervertebral disc. Absence of suitable graft tissue limits therapeutic approaches for repair of disc tissue. For this reason, there is considerable interest in developing "tissue engineering" strategies for the regeneration of the nucleus pulposus. Methods Rat mesenchymal stem cells were immobilized in 3-dimensional alginate hydrogels and cultured in a medium containing transforming growth factor-beta1 under hypoxia (2% O2) and normoxia (20% O2). Mesenchymal stem cells were examined by confocal microscopy to evaluate their viability and metabolic status after labeling with Celltracker green, a thiol sensitive dye, and Mitotracker red, a dye sensitive to the mitochondrial membrane potential. Flow cytometry, semiquantitative reverse transcription polymerase chain reaction and Western blot analysis were carried out to evaluate phenotypic and biosynthetic activities and the signaling pathways involved in the differentiation process. Results Under hypoxic conditions, mesenchymal stem cells formed large aggregates and exhibited positive Celltracker and Mitotracker signals. Glucose transporter-3, matrix metalloproteinase-2, collagen type II and type XI, and aggrecan mRNA and protein expression was upregulated, whereas there was no change in the levels of decorin, biglycan, fibromodulin, and lumican. Hypoxia maintained the expression of CD44 (hyaluronan receptor), ALCAM (CD166), and endoglin (transforming growth factor-beta receptor). Likewise, expression of beta3 and Alpha2 Integrin was upregulated. Transforming growth factor-beta treatment increased MAPK activity and Sox-9, aggrecan, and collagen type II gene expression. Basal levels of the phosphorylated MAPK isoform ERK1/2, but not p38, were higher under hypoxic conditions than normoxia, and its activation was further augmented by treatment of cells with transforming growth factor-beta. In hypoxia, transforming growth factor-beta sustained phosphorylated p38 expression for an extended time period. Pharmacological inhibition of ERK1/2 and p38 enzymatic activity resulted in a decrease in Sox-9, aggrecan, and collagen type II mRNA levels. Conclusions Our results indicate that hypoxia and transforming growth factor-beta drive mesenchymal stem cell differentiation towards a phenotype consistent with that of the nucleus pulposus. Measurement of selected signaling molecules and response to specific inhibitors suggest involvement of MAPK signaling pathways. It is concluded that mesenchymal stem cells could be used to repopulate the damaged or degenerate intervertebral disc.
Bernd Jilma - One of the best experts on this subject based on the ideXlab platform.
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Impaired Platelet Function among Platelet Donors
Thrombosis and Haemostasis, 2001Co-Authors: Petra Jilma-stohlawetz, Nicole Hergovich, Monika Homoncik, Larisa Dzirlo, Michaela Horvath, Michael Janisiw, Simon Panzer, Bernd JilmaAbstract:Summary Background: Platelet transfusions are effective for the prevention and treatment of bleeding in patients with disorders of platelet number and/or function. In recent years plateletpheresis concentrates have outnumbered pooled platelet concentrates, albeit with significant differences between nations. Thus, the platelet quality of individual donors has become increasingly important. The aim of this study was to gain an estimate for the prevalence of impaired platelet function among platelet donors. Study design and methods: We determined the inter-donor variability in platelet plug formation with a PFA-100 analyzer, the prevalence of impaired thromboxane formation, and effects of the density in Alpha2 Integrin polymorphism and density. Results: (i) Collagen-epinephrine induced closure time (CEPI-CT) showed a great inter-subject variability in platelet donors and was higher than in healthy controls (p = 0.008). One-fifth of donors had abnormal CEPI-CT values and 11% exceeded >300 s (max measurable value). (ii) Decreased serum thromboxane B2 levels were found in 9% of all donors, compatible with surreptitious intake of cyclooxygenase inhibitors or with an aspirin-like defect. CEPI-CT correlated inversely with TxB2-levels in donors and controls. (iii) The density of the Alpha2-Integrin correlated negatively with CEPI-CT and CADP-CT values in controls, but was not responsible for the observed impaired platelet function in donors. (iv) Finally, the ABO blood group system modulates closure times. Conclusion: In sum, a large number of platelet donors present with prolonged closure times. Decreased thromboxane formation and frequent platelet donation partly account for this observation. Abbreviations: 807 CC/CT/TT polymorphisms of the Alpha2-Integrin gene, CD36… GPIIIb, an alternative collagen receptor, CD42b… GpIb, the von Willebrand receptor, CD49b… Alpha2-Integrin, subunit of the main collagen receptor, CADP-CT… collagen adenosine diphosphate induced closure time, CEPI-CT… collagen/epinephrine induced closure time, PFA-100… platelet function analyzer, vWF-Ag… von Willebrand factor antigen, TXB2… thromboxane B2
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Impaired Platelet Function among Platelet Donors
Thrombosis and haemostasis, 2001Co-Authors: Petra Jilma-stohlawetz, Nicole Hergovich, Monika Homoncik, Larisa Dzirlo, Michaela Horvath, Michael Janisiw, Simon Panzer, Bernd JilmaAbstract:Background: Platelet transfusions are effective for the prevention and treatment of bleeding in patients with disorders of platelet number and/or function. In recent years plateletpheresis concentrates have outnumbered pooled platelet concentrates, albeit with significant differences between nations. Thus, the platelet quality of individual donors has become increasingly important. The aim of this study was to gain an estimate for the prevalence of impaired platelet function among platelet donors. Study design and methods:We determined the interdonor variability in platelet plug formation with a PFA-100 analyzer, the prevalence of impaired thromboxane formation, and effects of the density in Alpha2 Integrin polymorphism and density. Results: (i) Collagen-epinephrine induced closure time (CEPI-CT) showed a great inter-subject variability in platelet donors and was higher than in healthy controls (p = 0.008). One-fifth of donors had abnormal CEPI-CT values and 11% exceeded >300 s (max measurable value). (ii) Decreased serum thromboxane B2 levels were found in 9% of all donors, compatible with surreptitious intake of cyclooxygenase inhibitors or with an aspirin-like defect. CEPI-CT correlated inversely with TxB2-levels in donors and controls. (iii) The density of the Alpha2- Integrin correlated negatively with CEPI-CT and CADP-CT values in controls, but was not responsible for the observed impaired platelet function in donors. (iv) Finally, the ABO blood group system modulates closure times. Conclusion: In sum, a large number of platelet donors present with prolonged closure times. Decreased thromboxane formation and frequent platelet donation partly account for this observation.
Asha Guttapalli - One of the best experts on this subject based on the ideXlab platform.
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Hypoxia activates MAPK activity in rat nucleus pulposus cells: regulation of Integrin expression and cell survival.
Spine, 2005Co-Authors: Makarand V. Risbud, Asha Guttapalli, Todd J. Albert, Irving M. ShapiroAbstract:OBJECTIVE The aim of the present study was to investigate whether activation of MAPK subtypes ERK and p38 influences Integrin expression and promotes nucleus pulposus cell survival in hypoxia. SUMMARY OF BACKGROUND DATA We have recently shown that in a low oxygen environment, rat nucleus pulposus cells activate phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways. However, the role of these signaling pathways in regulating cell matrix interactions is not known. METHODS Rat nucleus pulposus cells were cultured in hypoxia (2% O2) or normoxia (20% O2). Western blotting and kinase assay were used to analyze expression of MAPK signaling molecules. Cell attachment to collagen was studied using an adhesion assay, whereas flow cytometry and RT-PCR were performed to quantify Integrin receptor expression. RESULTS In a hypoxic environment (2% O2), rat nucleus pulposus cells showed a persistent phosphorylation of p38 and ERK proteins; pERK catalyzed the phosphorylation of Elk1-Gst fusion protein. When ERK activity was blocked, cell adhesion to Type II collagen, one of the major extracellular matrix proteins in the nucleus pulposus tissue, was impaired. A similar inhibitory effect on collagen adhesion was observed when nucleus pulposus cells were treated with an antibody to Alpha2 Integrin. Furthermore, when ERK activity was inhibited, there was a decrease in Alpha2 Integrin mRNA expression. In contrast to ERK, inhibition of p38 activity did not modulate Alpha2 Integrin subunit mRNA expression. Likewise, inhibition of ERK, but not p38, resulted in downregulation of nucleus pulposus Alpha2 Integrin protein levels and blocked cell survival in hypoxia. CONCLUSIONS Hypoxia activated MAPK signaling pathway activity in nucleus pulposus cells. ERK, but not p38, regulated Alpha2 Integrin expression and cell survival.
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differentiation of mesenchymal stem cells towards a nucleus pulposus like phenotype in vitro implications for cell based transplantation therapy
Spine, 2004Co-Authors: Makarand V. Risbud, Asha Guttapalli, Todd J. Albert, Edward J Vresilovic, Alan S Hillibrand, Alexander R Vaccaro, Irving M. ShapiroAbstract:Objective Because mesenchymal stem cells can differentiate into chondrocyte-like cells, we ask the question, can mesenchymal stem cells commit to the nucleus pulposus phenotype? Background Back pain, a significant source of morbidity in our society, is linked to degenerative changes of the intervertebral disc. Absence of suitable graft tissue limits therapeutic approaches for repair of disc tissue. For this reason, there is considerable interest in developing "tissue engineering" strategies for the regeneration of the nucleus pulposus. Methods Rat mesenchymal stem cells were immobilized in 3-dimensional alginate hydrogels and cultured in a medium containing transforming growth factor-beta1 under hypoxia (2% O2) and normoxia (20% O2). Mesenchymal stem cells were examined by confocal microscopy to evaluate their viability and metabolic status after labeling with Celltracker green, a thiol sensitive dye, and Mitotracker red, a dye sensitive to the mitochondrial membrane potential. Flow cytometry, semiquantitative reverse transcription polymerase chain reaction and Western blot analysis were carried out to evaluate phenotypic and biosynthetic activities and the signaling pathways involved in the differentiation process. Results Under hypoxic conditions, mesenchymal stem cells formed large aggregates and exhibited positive Celltracker and Mitotracker signals. Glucose transporter-3, matrix metalloproteinase-2, collagen type II and type XI, and aggrecan mRNA and protein expression was upregulated, whereas there was no change in the levels of decorin, biglycan, fibromodulin, and lumican. Hypoxia maintained the expression of CD44 (hyaluronan receptor), ALCAM (CD166), and endoglin (transforming growth factor-beta receptor). Likewise, expression of beta3 and Alpha2 Integrin was upregulated. Transforming growth factor-beta treatment increased MAPK activity and Sox-9, aggrecan, and collagen type II gene expression. Basal levels of the phosphorylated MAPK isoform ERK1/2, but not p38, were higher under hypoxic conditions than normoxia, and its activation was further augmented by treatment of cells with transforming growth factor-beta. In hypoxia, transforming growth factor-beta sustained phosphorylated p38 expression for an extended time period. Pharmacological inhibition of ERK1/2 and p38 enzymatic activity resulted in a decrease in Sox-9, aggrecan, and collagen type II mRNA levels. Conclusions Our results indicate that hypoxia and transforming growth factor-beta drive mesenchymal stem cell differentiation towards a phenotype consistent with that of the nucleus pulposus. Measurement of selected signaling molecules and response to specific inhibitors suggest involvement of MAPK signaling pathways. It is concluded that mesenchymal stem cells could be used to repopulate the damaged or degenerate intervertebral disc.
Todd J. Albert - One of the best experts on this subject based on the ideXlab platform.
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Hypoxia activates MAPK activity in rat nucleus pulposus cells: regulation of Integrin expression and cell survival.
Spine, 2005Co-Authors: Makarand V. Risbud, Asha Guttapalli, Todd J. Albert, Irving M. ShapiroAbstract:OBJECTIVE The aim of the present study was to investigate whether activation of MAPK subtypes ERK and p38 influences Integrin expression and promotes nucleus pulposus cell survival in hypoxia. SUMMARY OF BACKGROUND DATA We have recently shown that in a low oxygen environment, rat nucleus pulposus cells activate phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways. However, the role of these signaling pathways in regulating cell matrix interactions is not known. METHODS Rat nucleus pulposus cells were cultured in hypoxia (2% O2) or normoxia (20% O2). Western blotting and kinase assay were used to analyze expression of MAPK signaling molecules. Cell attachment to collagen was studied using an adhesion assay, whereas flow cytometry and RT-PCR were performed to quantify Integrin receptor expression. RESULTS In a hypoxic environment (2% O2), rat nucleus pulposus cells showed a persistent phosphorylation of p38 and ERK proteins; pERK catalyzed the phosphorylation of Elk1-Gst fusion protein. When ERK activity was blocked, cell adhesion to Type II collagen, one of the major extracellular matrix proteins in the nucleus pulposus tissue, was impaired. A similar inhibitory effect on collagen adhesion was observed when nucleus pulposus cells were treated with an antibody to Alpha2 Integrin. Furthermore, when ERK activity was inhibited, there was a decrease in Alpha2 Integrin mRNA expression. In contrast to ERK, inhibition of p38 activity did not modulate Alpha2 Integrin subunit mRNA expression. Likewise, inhibition of ERK, but not p38, resulted in downregulation of nucleus pulposus Alpha2 Integrin protein levels and blocked cell survival in hypoxia. CONCLUSIONS Hypoxia activated MAPK signaling pathway activity in nucleus pulposus cells. ERK, but not p38, regulated Alpha2 Integrin expression and cell survival.
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differentiation of mesenchymal stem cells towards a nucleus pulposus like phenotype in vitro implications for cell based transplantation therapy
Spine, 2004Co-Authors: Makarand V. Risbud, Asha Guttapalli, Todd J. Albert, Edward J Vresilovic, Alan S Hillibrand, Alexander R Vaccaro, Irving M. ShapiroAbstract:Objective Because mesenchymal stem cells can differentiate into chondrocyte-like cells, we ask the question, can mesenchymal stem cells commit to the nucleus pulposus phenotype? Background Back pain, a significant source of morbidity in our society, is linked to degenerative changes of the intervertebral disc. Absence of suitable graft tissue limits therapeutic approaches for repair of disc tissue. For this reason, there is considerable interest in developing "tissue engineering" strategies for the regeneration of the nucleus pulposus. Methods Rat mesenchymal stem cells were immobilized in 3-dimensional alginate hydrogels and cultured in a medium containing transforming growth factor-beta1 under hypoxia (2% O2) and normoxia (20% O2). Mesenchymal stem cells were examined by confocal microscopy to evaluate their viability and metabolic status after labeling with Celltracker green, a thiol sensitive dye, and Mitotracker red, a dye sensitive to the mitochondrial membrane potential. Flow cytometry, semiquantitative reverse transcription polymerase chain reaction and Western blot analysis were carried out to evaluate phenotypic and biosynthetic activities and the signaling pathways involved in the differentiation process. Results Under hypoxic conditions, mesenchymal stem cells formed large aggregates and exhibited positive Celltracker and Mitotracker signals. Glucose transporter-3, matrix metalloproteinase-2, collagen type II and type XI, and aggrecan mRNA and protein expression was upregulated, whereas there was no change in the levels of decorin, biglycan, fibromodulin, and lumican. Hypoxia maintained the expression of CD44 (hyaluronan receptor), ALCAM (CD166), and endoglin (transforming growth factor-beta receptor). Likewise, expression of beta3 and Alpha2 Integrin was upregulated. Transforming growth factor-beta treatment increased MAPK activity and Sox-9, aggrecan, and collagen type II gene expression. Basal levels of the phosphorylated MAPK isoform ERK1/2, but not p38, were higher under hypoxic conditions than normoxia, and its activation was further augmented by treatment of cells with transforming growth factor-beta. In hypoxia, transforming growth factor-beta sustained phosphorylated p38 expression for an extended time period. Pharmacological inhibition of ERK1/2 and p38 enzymatic activity resulted in a decrease in Sox-9, aggrecan, and collagen type II mRNA levels. Conclusions Our results indicate that hypoxia and transforming growth factor-beta drive mesenchymal stem cell differentiation towards a phenotype consistent with that of the nucleus pulposus. Measurement of selected signaling molecules and response to specific inhibitors suggest involvement of MAPK signaling pathways. It is concluded that mesenchymal stem cells could be used to repopulate the damaged or degenerate intervertebral disc.