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Zhu K, Zhao R, Ye Y, Xu G, Zhang C. Effect of lentivirus-mediated growth and differentiation factor-5 transfection on differentiation of rabbit nucleus pulposus mesenchymal stem cells. Eur J Med Res 2022; 27:5. [PMID: 35022077 PMCID: PMC8756615 DOI: 10.1186/s40001-021-00624-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2020] [Accepted: 12/14/2021] [Indexed: 11/24/2022] Open
Abstract
Background Intervertebral disc degeneration (IDD) is a natural progression of age-related processes. Associated with IDD, degenerative disc disease (DDD) is a pathologic condition implicated as a major cause of chronic lower back pain, which can have a severe impact on the quality of life of patients. As degeneration progression is associated with elevated levels of inflammatory cytokines, enhanced aggrecan and collagen degradation, and changes in the disc cell phenotype. The purpose of this study was to investigate the biological and cytological characteristics of rabbit nucleus pulposus mesenchymal stem cells (NPMSCs)—a key factor in IDD—and to determine the effect of the growth and differentiation factor-5 (GDF5) on the differentiation of rabbit NPMSCs transduced with a lentivirus vector. Methods An in vitro culture model of rabbit NPMSCs was established and NPMSCs were identified by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR). Subsequently, NPMSCs were randomly divided into three groups: a transfection group (the lentiviral vector carrying GDF5 gene used to transfect NPMSCs); a control virus group (the NPMSCs transfected with an ordinary lentiviral vector); and a normal group (the NPMSCs alone). FCM, qRT-PCR, and western blot (WB) were used to detect the changes in NPMSCs. Results The GDF5-transfected NPMSCs displayed an elongated shape, with decreased cell density, and significantly increased GDF5 positivity rate in the transfected group compared to the other two groups (P < 0.01). The mRNA levels of Krt8, Krt18, and Krt19 in the transfected group were significantly higher in comparison with the other two groups (P < 0.01), and the WB results were consistent with that of qRT-PCR. Conclusions GDF5 could induce the differentiation of NPMSCs. The lentiviral vector carrying the GDF5 gene could be integrated into the chromosome genome of NPMSCs and promoted differentiation of NPMSCs into nucleus pulposus cells. Our findings advance the development of feasible and effective therapies for IDD. Supplementary Information The online version contains supplementary material available at 10.1186/s40001-021-00624-5.
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Affiliation(s)
- Kun Zhu
- Department of Orthopaedics, The First Affiliated Hospital of Bengbu Medical College, No. 287, Changhuai Road, Bengbu, 233000, Anhui, China
| | - Rui Zhao
- Department of General Medicine, Bengbu Medical College, Bengbu, China
| | - Yuchen Ye
- Department of Orthopaedics, The First Affiliated Hospital of Bengbu Medical College, No. 287, Changhuai Road, Bengbu, 233000, Anhui, China
| | - Gang Xu
- Department of Orthopaedics, The First Affiliated Hospital of Bengbu Medical College, No. 287, Changhuai Road, Bengbu, 233000, Anhui, China.
| | - Changchun Zhang
- Department of Orthopaedics, The First Affiliated Hospital of Bengbu Medical College, No. 287, Changhuai Road, Bengbu, 233000, Anhui, China.
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Guerrero J, Häckel S, Croft AS, Albers CE, Gantenbein B. The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency. JOR Spine 2021; 4:e1131. [PMID: 33778405 PMCID: PMC7984018 DOI: 10.1002/jsp2.1131] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Revised: 10/29/2020] [Accepted: 11/04/2020] [Indexed: 02/06/2023] Open
Abstract
INTRODUCTION Low back pain (LBP) is a global health concern. Increasing evidence implicates intervertebral disk (IVD) degeneration as a major contributor. In this respect, tissue-specific progenitors may play a crucial role in tissue regeneration, as these cells are perfectly adapted to their niche. Recently, a novel progenitor cell population was described in the nucleus pulposus (NP) that is positive for Tie2 marker. These cells have self-renewal capacity and in vitro multipotency potential. However, extremely low numbers of the NP progenitors limit the feasibility of cell therapy strategies. OBJECTIVE Here, we studied the influence of the culture method and of the microenvironment on the proliferation rate and the differentiation potential of human NP progenitors in vitro. METHOD Cells were obtained from human NP tissue from trauma patients. Briefly, the NP tissue cells were cultured in two-dimensional (2D) (monolayer) or three-dimensional (3D) (alginate beads) conditions. After 1 week, cells from 2D or 3D culture were expanded on fibronectin-coated flasks. Subsequently, expanded NP cells were then characterized by cytometry and tri-lineage differentiation, which was analyzed by qPCR and histology. Moreover, experiments using Tie2+ and Tie2- NP cells were also performed. RESULTS The present study aims to demonstrate that 3D expansion of NP cells better preserves the Tie2+ cell populations and increases the chondrogenic and osteogenic differentiation potential compared to 2D expansion. Moreover, the cell sorting experiments reveal that only Tie2+ cells were able to maintain the pluripotent gene expression if cultured in 3D within alginate beads. Therefore, our results highly suggest that the maintenance of the cell's multipotency is mainly, but not exclusively, due to the higher presence of Tie2+ cells due to 3D culture. CONCLUSION This project not only might have a scientific impact by evaluating the influence of a two-step expansion protocol on the functionality of NP progenitors, but it could also lead to an innovative clinical approach.
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Affiliation(s)
- Julien Guerrero
- Tissue Engineering for Orthopaedics & Mechanobiology, Department for BioMedical Research (DBMR) of the Faculty of Medicine of the University of BernUniversity of BernSwitzerland
| | - Sonja Häckel
- Department of Orthopaedic Surgery & Traumatology, InselspitalBern University HospitalBernSwitzerland
| | - Andreas S. Croft
- Tissue Engineering for Orthopaedics & Mechanobiology, Department for BioMedical Research (DBMR) of the Faculty of Medicine of the University of BernUniversity of BernSwitzerland
| | - Christoph E. Albers
- Department of Orthopaedic Surgery & Traumatology, InselspitalBern University HospitalBernSwitzerland
| | - Benjamin Gantenbein
- Tissue Engineering for Orthopaedics & Mechanobiology, Department for BioMedical Research (DBMR) of the Faculty of Medicine of the University of BernUniversity of BernSwitzerland
- Department of Orthopaedic Surgery & Traumatology, InselspitalBern University HospitalBernSwitzerland
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Li Z, Chen S, Ma K, He R, Xiong L, Hu Y, Deng X, Yang A, Ma X, Shao Z. Comparison of different methods for the isolation and purification of rat nucleus pulposus-derived mesenchymal stem cells. Connect Tissue Res 2020; 61:426-434. [PMID: 31203667 DOI: 10.1080/03008207.2019.1611793] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
PURPOSE Recently, nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have been identified and have shown good prospects for the repair of degenerative intervertebral discs. However, there is no consensus about the methods for the isolation and purification of NPMSCs. Therefore, a reliable and efficient isolation and purification method is potentially needed. We aimed to compare different methods and to identify an optimal method for isolating and purifying NPMSCs. METHODS NPMSCs were isolated and purified using two common methods (a low-density culture (LD) method and a mesenchymal stem cell complete medium culture (MSC-CM) method) and two novel methods (a cloning cylinder (CC) method and a combination of the CC and MSC-CM methods (MSC-CM+CC)). The morphology, MSC-specific surface markers (CD44, CD73, CD90, CD105, CD34 and HLA-DR), multiple-lineage differentiation potential, colony formation ability, and stemness gene (Oct4, Nanog, and Sox2) expression were evaluated and compared. RESULTS NPMSCs isolated from nucleus pulposus (NP) tissues via the four methods met the criteria stated by the International Society of Cell Therapy (ISCT) for MSCs, including adherent growth ability, MSC-specific surface antigen expression, and multi-lineage differentiation potential. In particular, the MSC-CM+CC method yielded a relatively higher quality of NPMSCs in terms of cell surface markers, multiple-lineage differentiation potential, colony formation ability, and stemness gene expression. CONCLUSIONS Our results indicated that NPMSCs can be obtained via all four methods and that the MSC-CM+CC method is more reliable and efficient than the other three methods. The findings from this study provide an alternative option for isolating and purifying NPMSCs.
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Affiliation(s)
- Zhiliang Li
- Department of Orthopedics, Renmin Hospital of Wuhan University , Wuhan, China
| | - Sheng Chen
- Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, China
| | - Kaige Ma
- Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, China
| | - Ruijun He
- Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, China
| | - Liming Xiong
- Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, China
| | - Yiqiang Hu
- Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, China
| | - Xiangyu Deng
- Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, China
| | - Aoxue Yang
- Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, China
| | - Xuan Ma
- Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, China
| | - Zengwu Shao
- Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan, China
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Liu Y, Li Y, Nan LP, Wang F, Zhou SF, Feng XM, Liu H, Zhang L. Insights of stem cell-based endogenous repair of intervertebral disc degeneration. World J Stem Cells 2020; 12:266-276. [PMID: 32399135 PMCID: PMC7202923 DOI: 10.4252/wjsc.v12.i4.266] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/01/2020] [Revised: 03/26/2020] [Accepted: 04/05/2020] [Indexed: 02/06/2023] Open
Abstract
Low back pain has become more prevalent in recent years, causing enormous economic burden for society and government. Common therapies used in clinics including conservative treatment and surgery can only relieve pain. Subsequent cell-based treatment such as mesenchymal stem cell transplantation poses problems such as short duration of therapeutic effect and tumorigenesis. Recently, the discovery and identification of stem cell niche and stem/progenitor cells in intervertebral disc bring increased attention to endogenous repair strategy. Therefore, we review the studies involving endogenous repair strategy and present the characteristics and current status of this treatment. Meanwhile, we also discuss the strategy and perspective of endogenous repair strategy in future.
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Affiliation(s)
- Yang Liu
- Department of Orthopedics, West China Hospital of Sichuan University, Chengdu 610000, Sichuan Province, China
- Department of Orthopedics, Dalian Medical University, Dalian 116000, Liaoning Province, China
| | - Yan Li
- Department of Oncology, The Affiliated Cancer Hospital, School of Medicine, UESTC, Chengdu 610000, Sichuan Province, China
| | - Li-Ping Nan
- Department of Orthopedics, Dalian Medical University, Dalian 116000, Liaoning Province, China
| | - Feng Wang
- Department of Orthopedics, Dalian Medical University, Dalian 116000, Liaoning Province, China
| | - Shi-Feng Zhou
- Department of Orthopedics, Clinical Medical College of Yangzhou University, Yangzhou 225000, Jiangsu Province, China
| | - Xin-Min Feng
- Department of Orthopedics, Clinical Medical College of Yangzhou University, Yangzhou 225000, Jiangsu Province, China
| | - Hao Liu
- Department of Orthopedics, West China Hospital of Sichuan University, Chengdu 610000, Sichuan Province, China
| | - Liang Zhang
- Department of Orthopedics, Clinical Medical College of Yangzhou University, Yangzhou 225000, Jiangsu Province, China
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Intervertebral Disc-Derived Stem/Progenitor Cells as a Promising Cell Source for Intervertebral Disc Regeneration. Stem Cells Int 2018; 2018:7412304. [PMID: 30662469 PMCID: PMC6312624 DOI: 10.1155/2018/7412304] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2018] [Revised: 10/18/2018] [Accepted: 11/05/2018] [Indexed: 12/14/2022] Open
Abstract
Intervertebral disc (IVD) degeneration is considered to be the primary reason for low back pain. Despite remarkable improvements in both pharmacological and surgical management of IVD degeneration (IVDD), therapeutic effects are still unsatisfactory. It is because of the fact that these therapies are mainly focused on alleviating the symptoms rather than treating the underlying cause or restoring the structure and biomechanical function of the IVD. Accumulating evidence has revealed that the endogenous stem/progenitor cells exist in the IVD, and these cells might be a promising cell source in the regeneration of degenerated IVD. However, the biological characteristics and potential application of IVD-derived stem/progenitor cells (IVDSCs) have yet to be investigated in detail. In this review, the authors aim to perform a review to systematically discuss (1) the isolation, surface markers, classification, and biological characteristics of IVDSCs; (2) the aging- and degeneration-related changes of IVDSCs and the influences of IVD microenvironment on IVDSCs; and (3) the potential for IVDSCs to promote regeneration of degenerated IVD. The authors believe that this review exclusively address the current understanding of IVDSCs and provide a novel approach for the IVD regeneration.
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Icariin Improves the Viability and Function of Cryopreserved Human Nucleus Pulposus-Derived Mesenchymal Stem Cells. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2018; 2018:3459612. [PMID: 30050653 PMCID: PMC6040248 DOI: 10.1155/2018/3459612] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/11/2018] [Revised: 05/09/2018] [Accepted: 05/22/2018] [Indexed: 12/18/2022]
Abstract
Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have shown a good prospect in the regeneration of intervertebral disc (IVD) tissues. However, fresh NPMSCs are not always readily available for basic research and clinical applications. Therefore, there is a need for an effective long-term cryopreservation method for NPMSCs. The aim of this study was to determine whether adding icariin (ICA) to the conventional cryoprotectant containing dimethyl sulfoxide (DMSO) had a better cryoprotective effect for NPMSCs. The results showed that the freezing solution containing ICA along with DMSO significantly increased the postthawed cell viability, decreased the apoptosis rate, improved cell adherence, and maintained the mitochondrial functions, as compared to the freezing solution containing DMSO alone. And the inhibition of oxidative stress and upregulation of heat shock proteins (HSPs) in the presence of ICA also confirmed the beneficial effect of ICA. Furthermore, ICA had no cytotoxicity and did not alter the characteristics of postthawed NPMSCs. In conclusion, these results suggested that the addition of ICA to the conventional freezing medium could improve the viability and function of the cryopreserved human NPMSCs and provided an optimal formulated freezing solution for human NPMSC cryopreservation.
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