Logo Logo
Hilfe
Kontakt
Switch language to English
Investigation of the effect of low oxygen tension on the osteogenic differentiation of human mesenchymal stem cells
Investigation of the effect of low oxygen tension on the osteogenic differentiation of human mesenchymal stem cells
Osteogenic differentiation of hMSC into osteoblasts is a prerequisite for subsequent bone formation. Numerous studies have explored osteogenic differentiation under standard tissue culture conditions, which usually employ 21% of oxygen. However, bone precursor cells such as hMSC reside in stem cell niches of low oxygen atmospheres. Furthermore, they are subjected to low oxygen concentrations when cultured on three dimensional scaffolds in vitro for bone tissue engineering purposes, and even more so after transplantation when vascularisation has yet to be established. Similarly, hMSC are exposed to low oxygen in the fracture microenvironment following bony injury. Recent studies revealed that hypoxic preconditioning improves cellular engraftment and survival in low oxygen atmospheres. In the present study we therefore investigated the osteogenic differentiation potential of hMSC under 2% O2 (hypoxia) in comparison to a standard tissue culture oxygen atmosphere of 21% (normoxia). The success of differentiation was validated through Alizarin red staining and RT-PCR analysis of osteoblast markers ALP and OPN. We assessed osteogenic differentiation of hMSC following hypoxic preconditioning to address whether this pretreatment is beneficial for subsequent differentiation under low oxygen tension. To validate our findings we carefully characterised the extent of hypoxia exerted on cells with respect to cell survival (WST assay) and proliferation (growth curve). Furthermore we also tried to elucidate the role of HIF-1 alpha with respect to osteogenic differentiation under hypoxia via silencing RNA and DFO, a pharmacological agent. Finally we tested whether an immortalized hMSC-line (SCP-1) would serve as a model system for hMSC. We found that hMSC proliferate better if cultured under 2% of oxygen. We confirmed that osteogenic differentiation of hMSC is indeed inhibited under hypoxia. We showed for the first time that hypoxic preconditioning of hMSC prior to osteogenic induction restores osteogenic differentiation of hMSC under hypoxia. HIF-1 alpha seemed not to play a significant role in osteogenic differentiation under hypoxia, as transiently knocking down of HIF-1 alpha in preconditioned samples did not show any differences in their osteogenic differentiation. Moreover stabilising Hif-1 alpha in hypoxic samples did not yield any osteogenic differentiation either substantiating the notion that HIF-1 alpha does not have a direct role in the osteogenic differentiation of hMSC under hypoxia. Together our data suggest that hypoxia favours stemness over differentiation by upregulating embryonic stem cell markers like OCT-4 and NANOG. Hypoxic preconditioning may help to restore the otherwise reduced osteogenic potential of hMSC, either within a hypoxic fracture environment or at the site of implantation of tissue engineered bone constructs. We therefore believe that hypoxic preconditioning is a helpful tool for successful regenerative cell-based therapies in bone tissue engineering. SCP-1 cells might be used as a model system for hMSC as they are easy to handle, can be cultured to a desired cell number within a very short period of time, are relatively inexpensive and above all do not go into senescence as seen with hMSC after approximately 20 passages. Apart from their distinct advantages SCP-1 cells still maintain the specific CD markers characteristic for hMSC and are able to differentiate into adipogenic, osteogenic and chondrogenic lineages. However for in vivo experiments in animals a constant monitoring of neoplastic transformation is mandatory
Tissue engineering, bone, hypoxia, human mesenchymal stem cells, osteogenic differentiation, HIF-1 alpha
Cherian Kallukalam, Bobby
2010
Englisch
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Cherian Kallukalam, Bobby (2010): Investigation of the effect of low oxygen tension on the osteogenic differentiation of human mesenchymal stem cells. Dissertation, LMU München: Medizinische Fakultät
[thumbnail of Kallukalam_Cherian_Bobby.pdf]
Vorschau
PDF
Kallukalam_Cherian_Bobby.pdf

3MB

Abstract

Osteogenic differentiation of hMSC into osteoblasts is a prerequisite for subsequent bone formation. Numerous studies have explored osteogenic differentiation under standard tissue culture conditions, which usually employ 21% of oxygen. However, bone precursor cells such as hMSC reside in stem cell niches of low oxygen atmospheres. Furthermore, they are subjected to low oxygen concentrations when cultured on three dimensional scaffolds in vitro for bone tissue engineering purposes, and even more so after transplantation when vascularisation has yet to be established. Similarly, hMSC are exposed to low oxygen in the fracture microenvironment following bony injury. Recent studies revealed that hypoxic preconditioning improves cellular engraftment and survival in low oxygen atmospheres. In the present study we therefore investigated the osteogenic differentiation potential of hMSC under 2% O2 (hypoxia) in comparison to a standard tissue culture oxygen atmosphere of 21% (normoxia). The success of differentiation was validated through Alizarin red staining and RT-PCR analysis of osteoblast markers ALP and OPN. We assessed osteogenic differentiation of hMSC following hypoxic preconditioning to address whether this pretreatment is beneficial for subsequent differentiation under low oxygen tension. To validate our findings we carefully characterised the extent of hypoxia exerted on cells with respect to cell survival (WST assay) and proliferation (growth curve). Furthermore we also tried to elucidate the role of HIF-1 alpha with respect to osteogenic differentiation under hypoxia via silencing RNA and DFO, a pharmacological agent. Finally we tested whether an immortalized hMSC-line (SCP-1) would serve as a model system for hMSC. We found that hMSC proliferate better if cultured under 2% of oxygen. We confirmed that osteogenic differentiation of hMSC is indeed inhibited under hypoxia. We showed for the first time that hypoxic preconditioning of hMSC prior to osteogenic induction restores osteogenic differentiation of hMSC under hypoxia. HIF-1 alpha seemed not to play a significant role in osteogenic differentiation under hypoxia, as transiently knocking down of HIF-1 alpha in preconditioned samples did not show any differences in their osteogenic differentiation. Moreover stabilising Hif-1 alpha in hypoxic samples did not yield any osteogenic differentiation either substantiating the notion that HIF-1 alpha does not have a direct role in the osteogenic differentiation of hMSC under hypoxia. Together our data suggest that hypoxia favours stemness over differentiation by upregulating embryonic stem cell markers like OCT-4 and NANOG. Hypoxic preconditioning may help to restore the otherwise reduced osteogenic potential of hMSC, either within a hypoxic fracture environment or at the site of implantation of tissue engineered bone constructs. We therefore believe that hypoxic preconditioning is a helpful tool for successful regenerative cell-based therapies in bone tissue engineering. SCP-1 cells might be used as a model system for hMSC as they are easy to handle, can be cultured to a desired cell number within a very short period of time, are relatively inexpensive and above all do not go into senescence as seen with hMSC after approximately 20 passages. Apart from their distinct advantages SCP-1 cells still maintain the specific CD markers characteristic for hMSC and are able to differentiate into adipogenic, osteogenic and chondrogenic lineages. However for in vivo experiments in animals a constant monitoring of neoplastic transformation is mandatory