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1
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Madsen EJ, Rhee S, Wahlsten M, Calabrese TC, Kohn DH. Dual-Functional Peptide DPI-VTK Promotes Mesenchymal Stem Cell Migration for Bone Regeneration. J Biomed Mater Res A 2025; 113:e37908. [PMID: 40186383 PMCID: PMC11991734 DOI: 10.1002/jbm.a.37908] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2025] [Revised: 03/23/2025] [Accepted: 03/25/2025] [Indexed: 04/07/2025]
Abstract
Targeting specific populations of host cells with chemotactic and adhesion factors is a promising strategy for inducing bone regeneration without the use of exogenous cells. Two peptide sequences have been derived from phage display: the mesenchymal stem cell (MSC) binding DPI (DPIYALSWSGMA) sequence and the apatite binding VTK (VTKHLNQISQSY) sequence. When combined into the dual-functional sequence, DPI-VTK increases the adhesion strength of MSCs to apatite surfaces and the amount of bone formation with transplanted MSCs. Because many adhesion molecules can stimulate chemotaxis, and cell adhesion to peptide DPI-VTK is mediated by integrins also critical to migration, we hypothesized that DPI-VTK serves as an MSC-specific chemotactic factor and can increase bone regeneration by promoting the osteogenesis of the migrated host MSCs in vivo. In transwell assays, induced pluripotent stem cell-derived human MSCs (p < 0.0001) and primary mouse calvarial cells (p < 0.0001) showed significantly increased migration in vitro when DPI-VTK was used as a chemoattractant. Further characterization of DPI-VTK binding cells from mouse calvaria using flow cytometry showed specificity toward cells expressing MSC markers (CD29, CD73, CD90, CD105, CD106, Sca-1, CD44, and CD200). When conjugated to a mineralized scaffold in vivo, DPI-VTK increased the migration of CD90 and CD200 positive cells (p < 0.05) and increased bone formation versus no-peptide controls (p < 0.05). These results demonstrate the utility of phage display in creating multifunctional peptides that can increase migration, adhesion, and bone formation in vivo, a strategy that could be applied to numerous different cell types and systems. Results advance biomaterials-based bone regeneration in two ways-demonstrating the ability of the phage-derived peptides to increase the migration of MSCs in vivo and increase host-mediated bone regeneration-potentially bypassing cell transplantation.
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Affiliation(s)
- Eric J Madsen
- Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, Michigan
| | - Seungmeen Rhee
- Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, Michigan
| | - Madison Wahlsten
- Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, Michigan
- Department of Biomedical Engineering, University of Michigan, Ann Arbor Michigan
| | - Tia C Calabrese
- Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, Michigan
- Department of Biomedical Engineering, University of Michigan, Ann Arbor Michigan
| | - David H Kohn
- Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, Michigan
- Department of Biomedical Engineering, University of Michigan, Ann Arbor Michigan
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2
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Gu C, Tang Q, Li L, Chen Y. Optimization and Implication of Adipose-Derived Stem Cells in Craniofacial Bone Regeneration and Repair. Bioengineering (Basel) 2024; 11:1100. [PMID: 39593759 PMCID: PMC11592193 DOI: 10.3390/bioengineering11111100] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2024] [Revised: 10/17/2024] [Accepted: 10/27/2024] [Indexed: 11/28/2024] Open
Abstract
Adipose-derived stem cells (ADSCs) have emerged as a promising resource for craniofacial bone regeneration due to their high abundance and easy accessibility, significant osteogenic potential, versatile applications, and potential for personalized medicine, which underscore their importance in this field. This article reviews the current progress of preclinical studies that describe the careful selection of specific ADSC subpopulations, key signaling pathways involved, and usage of various strategies to enhance the osteogenic potential of ADSCs. Additionally, clinical case reports regarding the application of ADSCs in the repair of calvarial defects, cranio-maxillofacial defects, and alveolar bone defects are also discussed.
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Affiliation(s)
- Cong Gu
- Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118, USA; (Q.T.); (L.L.); (Y.C.)
| | - Qinghuang Tang
- Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118, USA; (Q.T.); (L.L.); (Y.C.)
- Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, NY 14214, USA
| | - Liwen Li
- Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118, USA; (Q.T.); (L.L.); (Y.C.)
- Department of Biological Sciences, University at Buffalo, Buffalo, NY 14260, USA
| | - YiPing Chen
- Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118, USA; (Q.T.); (L.L.); (Y.C.)
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3
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Silva-Carvalho AÉ, Bispo ECI, da Silva IGM, Correa JR, Carvalho JL, Gelfuso GM, Saldanha-Araujo F. Characterization of ibrutinib's effects on the morphology, proliferation, phenotype, viability, and anti-inflammatory potential of adipose-derived mesenchymal stromal cells. Sci Rep 2024; 14:19906. [PMID: 39191849 DOI: 10.1038/s41598-024-71054-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2024] [Accepted: 08/23/2024] [Indexed: 08/29/2024] Open
Abstract
Ibrutinib (IB) is a tyrosine kinase inhibitor (TKI) that has immunomodulatory action and can be used as second-line therapy for steroid-refractory or steroid-resistant chronic Graft versus Host Disease (cGVHD). Mesenchymal stromal cells (MSCs) are distributed throughout the body and their infusion has also been explored as a second-line therapeutic alternative for the treatment of cGVHD. Considering the currently unknown effects of IB on endogenous MSCs, as well as the possible combined use of IB and MSCs for cGVHD, we investigated whether adipose tissue-derived MSCs present IB-targets, as well as the consequences of treating MSCs with this drug, regarding cell viability, proliferation, phenotype, and anti-inflammatory potential. Interestingly, we show for the first time that MSCs express several IB target genes. Also of note, the treatment of such cells with this TKI elevated the levels of CD90 and CD105 surface proteins, as well as VCAM-1. Furthermore, IB-treated MSCs presented increased mRNA expression of the anti-inflammatory genes PD-L1, TSG-6, and IL-10. However, continued exposure to IB, even at low doses, compromised the viability of MSCs. These data indicate that the use of IB can stimulate an anti-inflammatory profile in MSCs, but also that a continued exposure to IB can compromise MSC viability over time.
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Affiliation(s)
- Amandda Évelin Silva-Carvalho
- Laboratório de Hematologia E Células-Tronco, Departamento de Ciências da Saúde, Universidade de Brasília, Campus Darcy Ribeiro, Brasília, DF, Brasil
- Laboratório de Farmacologia Molecular, Universidade de Brasília, Brasília, Brasil
| | - Elizabete Cristina Iseke Bispo
- Laboratório de Hematologia E Células-Tronco, Departamento de Ciências da Saúde, Universidade de Brasília, Campus Darcy Ribeiro, Brasília, DF, Brasil
| | | | - José Raimundo Correa
- Laboratório de Microscopia E Microanálises, Universidade de Brasília, Brasília, Brasil
| | - Juliana Lott Carvalho
- Laboratório Multidisciplinar de Biociências, Universidade de Brasília, Brasília, Brasil
| | | | - Felipe Saldanha-Araujo
- Laboratório de Hematologia E Células-Tronco, Departamento de Ciências da Saúde, Universidade de Brasília, Campus Darcy Ribeiro, Brasília, DF, Brasil.
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4
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Lee MY, Yoon HW, Lee SY, Kim KM, Shin SJ, Kwon JS. Mineral trioxide aggregate in membrane form as a barrier membrane in guided bone regeneration. J Dent Sci 2024; 19:1653-1666. [PMID: 39035317 PMCID: PMC11259731 DOI: 10.1016/j.jds.2023.11.021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2023] [Revised: 11/27/2023] [Indexed: 07/23/2024] Open
Abstract
Background/purpose In the field of conservative dentistry and endodontics, mineral trioxide aggregate (MTA), commonly used, possesses advantages such as biocompatibility, antimicrobial properties and osteogenic potential. This study investigated the feasibility of utilizing membrane form mineral trioxide aggregate (MTA) as a barrier membrane in guided bone regeneration (GBR) procedures. Materials and methods Membranes were electrospun from three different formulations: 15 w/v% Polycaprolactone (PCL), 13 w/v% PCL + 2 w/v% MTA (2MTA), and 11 w/v% PCL + 4 w/v% MTA (4MTA). Physicochemical and mechanical properties of the electrospun membrane were compared, encompassing parameters such as surface morphology, fiber diameter distribution, chemical composition, phase identification, tensile stress, pH variation, and water contact angle. Moreover, the antimicrobial properties against of the electrospun membranes were assessed through direct exposure to streptococcus aureus (S. aureus) and candida albicans (C. albicans). Additionally, on the 7th day, biocompatibility and cell attachment were investigated with respect to L929 (fibroblast) and MC3T3 (pre-osteoblast) cells. Inhibition of L929 cell infiltration and the expression of osteogenic related genes including osteocalcin (OCN), alkaline phosphatase (ALP), and runt related transcription factor 2 (RUNX2) in MC3T3 cells on 7th and 14th days were also investigated. Results PCL, 2MTA, and 4MTA exhibited no statistically differences in fiber diameter distribution and tensile stress. However, as the MTA content increased, wettability and pH also increased. Due to the elevated pH, 4MTA demonstrated the lowest viability S.aureus and C.albicans. All membranes were highly biocompatibility and promoted cell attachment, while effectively preventing L929 cell infiltration. Lastly 4MTA showed increase in OCN, ALP, and RUNX2 expression on both 7th and 14th day. Conclusion The membrane form MTA possessed characteristics essential for a novel barrier membrane.
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Affiliation(s)
- Min-Yong Lee
- Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of Dentistry, Seoul, South Korea
| | - Hi-Won Yoon
- Department of Conservative Dentistry, Gangnam Severance Hospital, Yonsei University College of Dentistry, Seoul, South Korea
| | - Si-Yoon Lee
- Department of Biology, New York University, New York, NY, USA
| | - Kwang-Mahn Kim
- Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of Dentistry, Seoul, South Korea
| | - Su-Jung Shin
- Department of Conservative Dentistry, Gangnam Severance Hospital, Yonsei University College of Dentistry, Seoul, South Korea
| | - Jae-Sung Kwon
- Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of Dentistry, Seoul, South Korea
- BK21 FOUR Project, Yonsei University College of Dentistry, Seoul, South Korea
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Morawska-Kozłowska M, Wilkosz A, Zhalniarovich Y. The Omentum-A Forgotten Structure in Veterinary Surgery in Small Animals' Surgery. Animals (Basel) 2024; 14:1848. [PMID: 38997960 PMCID: PMC11240631 DOI: 10.3390/ani14131848] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2024] [Revised: 06/16/2024] [Accepted: 06/17/2024] [Indexed: 07/14/2024] Open
Abstract
The greater and lesser omentum are derived from embryonic mesogastrium. The expansive greater omentum in dogs covers intestinal coils, while in cats, it is smaller. Comprising distinct portions, the greater omentum is rich in lymphatics and blood vessels. Conversely, the lesser omentum spans the liver, stomach, and duodenum. Studies on canine omentum reveal unique immune cell composition and regenerative potential attributed to adipose tissue-derived stromal cells (ADSCs). These cells hold promise in regenerative medicine, showing enhanced abilities compared with ADSCs from other sources. The omentum is critical in tissue repair and pathology, making it invaluable in veterinary surgery across various medical fields. The aim of this article was to research current knowledge about the applications of the omentum in veterinary surgery and the possibilities of using this structure in the future.
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Affiliation(s)
- Magdalena Morawska-Kozłowska
- Department of Surgery and Radiology with Clinic, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland
| | - Aleksandra Wilkosz
- Department of Surgery and Radiology with Clinic, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland
| | - Yauheni Zhalniarovich
- Department of Surgery and Radiology with Clinic, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland
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6
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Jinsheng L, Qing D, Junhao C, Qiqi S, Jieru C, Liwen Y, Zhiyun G, Tailin G, Jie W. Micro/nano topological modification of TiO 2 nanotubes activates Thy-1 signaling to control osteogenic differentiation of stem cells. SLAS DISCOVERY : ADVANCING LIFE SCIENCES R & D 2024; 29:100139. [PMID: 38169172 DOI: 10.1016/j.slasd.2023.12.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/18/2023] [Revised: 12/04/2023] [Accepted: 12/30/2023] [Indexed: 01/05/2024]
Abstract
Micro/nano topological modification is critical for improving the in vivo behaviors of bone implants, regulating multiple cellular functions. Titania (TiO2) nanotubes show the capacity of promoting osteoblast-related cell differentiation and induce effective osseointegration, serving as a model material for studying the effects of micro/nano-topological modifications on cells. However, the intracellular signaling pathways by which TiO2 nanotubes regulate the osteogenic differentiation of stem cells are not fully defined. Thy-1 (CD90), a cell surface glycoprotein anchored by glycosylphosphatidylinositol, has been considered a key molecule in osteoblast differentiation in recent years. Nevertheless, whether the micro/nano topology of the implant surface leads to changes in Thy-1 is unknown, as well as whether these changes promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Here, TiO2 nanotubes of various diameters were prepared by adjusting the anodizing voltage. qPCR and immunoblot were carried out to assess the mechanism by which TiO2 nanotubes regulate Thy-1. The results revealed Ti plates harboring TiO2 nanotubes ∼100-nm diameter (TNT-100) markedly upregulated Thy-1. Subsequently, upregulated Thy-1 promoted the activation of Fyn/RhoA/MLC Ⅱ/F-actin axis, which enhanced the nuclear translocation of YAP. After Thy-1 knockdown by siRNA, the Fyn/RhoA/MLC Ⅱ/F-actin axis was significantly inhibited and TiO2 nanotubes showed decreased effects on osteogenic differentiation. Therefore, Thy-1 upregulation might be a major mechanism by which micro/nano-topological modification of TiO2 nanotubes promotes osteogenic differentiation in BMSCs. This study provides novel insights into the molecular mechanism of TiO2 nanotubes, which may help design improved bone implants for clinical application.
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Affiliation(s)
- Li Jinsheng
- Institute of Biomedical Engineering, College of Medicine, Southwest Jiaotong University, Chengdu 610031, Sichuan, China; Key Laboratory of Advanced Technologies of Materials Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China
| | - Deng Qing
- Institute of Biomedical Engineering, College of Medicine, Southwest Jiaotong University, Chengdu 610031, Sichuan, China
| | - Chen Junhao
- School of Finance and Economics, Xizang Minzu University, Xianyang 712082, PR China
| | - Si Qiqi
- School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China
| | - Chen Jieru
- School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China
| | - Yang Liwen
- Institute of Biomedical Engineering, College of Medicine, Southwest Jiaotong University, Chengdu 610031, Sichuan, China; Key Laboratory of Advanced Technologies of Materials Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China
| | - Guo Zhiyun
- School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China
| | - Guo Tailin
- Institute of Biomedical Engineering, College of Medicine, Southwest Jiaotong University, Chengdu 610031, Sichuan, China; Key Laboratory of Advanced Technologies of Materials Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China.
| | - Weng Jie
- Institute of Biomedical Engineering, College of Medicine, Southwest Jiaotong University, Chengdu 610031, Sichuan, China; Key Laboratory of Advanced Technologies of Materials Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China.
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7
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Ferreira-Baptista C, Ferreira R, Fernandes MH, Gomes PS, Colaço B. Influence of the Anatomical Site on Adipose Tissue-Derived Stromal Cells' Biological Profile and Osteogenic Potential in Companion Animals. Vet Sci 2023; 10:673. [PMID: 38133224 PMCID: PMC10747344 DOI: 10.3390/vetsci10120673] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2023] [Revised: 11/13/2023] [Accepted: 11/20/2023] [Indexed: 12/23/2023] Open
Abstract
Adipose tissue-derived stromal cells (ADSCs) have generated considerable interest in the field of veterinary medicine, particularly for their potential in therapeutic strategies focused on bone regeneration. These cells possess unique biological characteristics, including their regenerative capacity and their ability to produce bioactive molecules. However, it is crucial to recognize that the characteristics of ADSCs can vary depending on the animal species and the site from which they are derived, such as the subcutaneous and visceral regions (SCAT and VAT, respectively). Thus, the present work aimed to comprehensively review the different traits of ADSCs isolated from diverse anatomical sites in companion animals, i.e., dogs, cats, and horses, in terms of immunophenotype, morphology, proliferation, and osteogenic differentiation potential. The findings indicate that the immunophenotype, proliferation, and osteogenic potential of ADSCs differ according to tissue origin and species. Generally, the proliferation rate is higher in VAT-derived ADSCs in dogs and horses, whereas in cats, the proliferation rate appears to be similar in both cells isolated from SCAT and VAT regions. In terms of osteogenic differentiation potential, VAT-derived ADSCs demonstrate the highest capability in cats, whereas SCAT-derived ADSCs exhibit superior potential in horses. Interestingly, in dogs, VAT-derived cells appear to have greater potential than those isolated from SCAT. Within the VAT, ADSCs derived from the falciform ligament and omentum show increased osteogenic potential, compared to cells isolated from other anatomical locations. Consequently, considering these disparities, optimizing isolation protocols becomes pivotal, tailoring them to the specific target species and therapeutic aims, and judiciously selecting the anatomical site for ADSC isolation. This approach holds promise to enhance the efficacy of ADSCs-based bone regenerative therapies.
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Affiliation(s)
- Carla Ferreira-Baptista
- Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os-Montes e Alto Douro (UTAD), 5000-801 Vila Real, Portugal;
- BoneLab—Laboratory for Bone Metabolism and Regeneration, Faculty of Dental Medicine, University of Porto, 4200-393 Porto, Portugal; (M.H.F.); (P.S.G.)
- REQUIMTE/LAQV, University of Porto, 4100-007 Porto, Portugal
- REQUIMTE/LAQV, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal;
| | - Rita Ferreira
- REQUIMTE/LAQV, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal;
| | - Maria Helena Fernandes
- BoneLab—Laboratory for Bone Metabolism and Regeneration, Faculty of Dental Medicine, University of Porto, 4200-393 Porto, Portugal; (M.H.F.); (P.S.G.)
- REQUIMTE/LAQV, University of Porto, 4100-007 Porto, Portugal
| | - Pedro Sousa Gomes
- BoneLab—Laboratory for Bone Metabolism and Regeneration, Faculty of Dental Medicine, University of Porto, 4200-393 Porto, Portugal; (M.H.F.); (P.S.G.)
- REQUIMTE/LAQV, University of Porto, 4100-007 Porto, Portugal
| | - Bruno Colaço
- Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os-Montes e Alto Douro (UTAD), 5000-801 Vila Real, Portugal;
- REQUIMTE/LAQV, University of Porto, 4100-007 Porto, Portugal
- CECAV—Animal and Veterinary Research Centre UTAD, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal
- Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), 5000-801 Vila Real, Portugal
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8
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Hoover MY, Ambrosi TH, Steininger HM, Koepke LS, Wang Y, Zhao L, Murphy MP, Alam AA, Arouge EJ, Butler MGK, Takematsu E, Stavitsky SP, Hu S, Sahoo D, Sinha R, Morri M, Neff N, Bishop J, Gardner M, Goodman S, Longaker M, Chan CKF. Purification and functional characterization of novel human skeletal stem cell lineages. Nat Protoc 2023; 18:2256-2282. [PMID: 37316563 PMCID: PMC10495180 DOI: 10.1038/s41596-023-00836-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2022] [Accepted: 03/21/2023] [Indexed: 06/16/2023]
Abstract
Human skeletal stem cells (hSSCs) hold tremendous therapeutic potential for developing new clinical strategies to effectively combat congenital and age-related musculoskeletal disorders. Unfortunately, refined methodologies for the proper isolation of bona fide hSSCs and the development of functional assays that accurately recapitulate their physiology within the skeleton have been lacking. Bone marrow-derived mesenchymal stromal cells (BMSCs), commonly used to describe the source of precursors for osteoblasts, chondrocytes, adipocytes and stroma, have held great promise as the basis of various approaches for cell therapy. However, the reproducibility and clinical efficacy of these attempts have been obscured by the heterogeneous nature of BMSCs due to their isolation by plastic adherence techniques. To address these limitations, our group has refined the purity of individual progenitor populations that are encompassed by BMSCs by identifying defined populations of bona fide hSSCs and their downstream progenitors that strictly give rise to skeletally restricted cell lineages. Here, we describe an advanced flow cytometric approach that utilizes an extensive panel of eight cell surface markers to define hSSCs; bone, cartilage and stromal progenitors; and more differentiated unipotent subtypes, including an osteogenic subset and three chondroprogenitors. We provide detailed instructions for the FACS-based isolation of hSSCs from various tissue sources, in vitro and in vivo skeletogenic functional assays, human xenograft mouse models and single-cell RNA sequencing analysis. This application of hSSC isolation can be performed by any researcher with basic skills in biology and flow cytometry within 1-2 days. The downstream functional assays can be performed within a range of 1-2 months.
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Affiliation(s)
- Malachia Y Hoover
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Thomas H Ambrosi
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
- Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA
- Department of Orthopaedic Surgery, UC Davis Health, Sacramento, CA, USA
| | - Holly M Steininger
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Lauren S Koepke
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Yuting Wang
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
- Department of Orthopaedic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Liming Zhao
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
- Department of Orthopaedic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Matthew P Murphy
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
- Blond McIndoe Laboratories, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, UK
| | - Alina A Alam
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Elizabeth J Arouge
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - M Gohazrua K Butler
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Eri Takematsu
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Suzan P Stavitsky
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Serena Hu
- Department of Orthopaedic Surgery, Stanford Hospitals and Clinics, Stanford, CA, USA
| | - Debashis Sahoo
- Department of Pathology, University of California San Diego, La Jolla, CA, USA
| | - Rahul Sinha
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Maurizio Morri
- Chan Zuckerberg BioHub, San Francisco, CA, USA
- Altos Labs, Redwood City, CA, USA
| | - Norma Neff
- Chan Zuckerberg BioHub, San Francisco, CA, USA
| | - Julius Bishop
- Department of Orthopaedic Surgery, Stanford Hospitals and Clinics, Stanford, CA, USA
| | - Michael Gardner
- Department of Orthopaedic Surgery, Stanford Hospitals and Clinics, Stanford, CA, USA
| | - Stuart Goodman
- Department of Orthopaedic Surgery, Stanford Hospitals and Clinics, Stanford, CA, USA
| | - Michael Longaker
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
- Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA
- Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School of Medicine, Stanford University, Stanford, CA, USA
| | - Charles K F Chan
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
- Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA.
- Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School of Medicine, Stanford University, Stanford, CA, USA.
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9
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Mulcrone PL, Lam AK, Frabutt D, Zhang J, Chrzanowski M, Herzog RW, Xiao W. Chemical modification of AAV9 capsid with N-ethyl maleimide alters vector tissue tropism. Sci Rep 2023; 13:8436. [PMID: 37231038 PMCID: PMC10212940 DOI: 10.1038/s41598-023-35547-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2023] [Accepted: 05/19/2023] [Indexed: 05/27/2023] Open
Abstract
Although more adeno-associated virus AAV-based drugs enter the clinic, vector tissue tropism remains an unresolved challenge that limits its full potential despite that the tissue tropism of naturally occurring AAV serotypes can be altered by genetic engineering capsid vie DNA shuffling, or molecular evolution. To further expand the tropism and thus potential applications of AAV vectors, we utilized an alternative approach that employs chemical modifications to covalently link small molecules to reactive exposed Lysine residues of AAV capsids. We demonstrated that AAV9 capsid modified with N-ethyl Maleimide (NEM) increased its tropism more towards murine bone marrow (osteoblast lineage) while decreased transduction of liver tissue compared to the unmodified capsid. In the bone marrow, AAV9-NEM transduced Cd31, Cd34, and Cd90 expressing cells at a higher percentage than unmodified AAV9. Moreover, AAV9-NEM localized strongly in vivo to cells lining the calcified trabecular bone and transduced primary murine osteoblasts in culture, while WT AAV9 transduced undifferentiated bone marrow stromal cells as well as osteoblasts. Our approach could provide a promising platform for expanding clinical AAV development to treat bone pathologies such as cancer and osteoporosis. Thus, chemical engineering the AAV capsid holds great potential for development of future generations of AAV vectors.
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Affiliation(s)
- Patrick L Mulcrone
- Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Anh K Lam
- Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Dylan Frabutt
- Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Junping Zhang
- Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Matthew Chrzanowski
- Lewis Katz School of Medicine, Temple University, Philadelphia, PA, 19140, USA
| | - Roland W Herzog
- Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Weidong Xiao
- Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
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10
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Das M, Teli P, Vaidya A, Kale V. Secretome of Young Mesenchymal Stromal Cells Rejuvenates Aged Mesenchymal Stromal Cells by Normalizing Their Phenotype and Restoring Their Differentiation Profile. Stem Cells Dev 2023; 32:12-24. [PMID: 36453235 DOI: 10.1089/scd.2022.0213] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022] Open
Abstract
During aging, the proliferation and differentiation ability of mesenchymal stem/stromal cells (MSCs) gets affected, and hence, aged MSCs are not preferred for regenerative purposes. Rapid identification of aging-associated changes within MSCs and the mechanistic pathways involved are necessary to determine optimal cell sources to treat musculoskeletal disorders in older patients. In the present study, we have identified a set of phenotypic markers, namely downregulated expression of CD90 and upregulated expression of CD45, as age-defining markers for the bone marrow-derived MSCs. We also show that these phenotypic changes in aged MSCs correlate with their aging-mediated differentiation defects. We find that oxidative stress signaling leading to the activation of nuclear factor kappa B (NF-κB) plays an essential role in altering the phenotype and differentiation ability of the aged MSCs. We further show that treatment of aged MSCs with the conditioned medium (CM) derived from young MSCs (young-CM) restored their phenotype and differentiation potential to the young-like by ameliorating activation of NF-κB signaling in them. Similar changes could also be achieved by using an inhibitor of NF-κB signaling, showing that oxidative stress-induced NF-κB activation is the causative factor in the aging of MSCs. Additionally, we show that treating young MSCs with hydrogen peroxide mimics all the aging-mediated changes in them, underscoring the involvement of oxidative stress in the aging of MSCs. Overall, our data suggest that the altered expression of CD90 and CD45 surface markers can be used as a primary screen to identify the onset of aging in the MSCs, which can be quickly reversed by their in vitro treatment with young-CM or NF-κB inhibitor. Our study also puts the phenotypic characterization of MSCs in a clinical perspective.
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Affiliation(s)
- Madhurima Das
- Symbiosis Centre for Stem Cell Research, Symbiosis International (Deemed University), Pune, India
| | - Prajakta Teli
- Symbiosis Centre for Stem Cell Research, Symbiosis International (Deemed University), Pune, India
| | - Anuradha Vaidya
- Symbiosis Centre for Stem Cell Research, Symbiosis International (Deemed University), Pune, India.,Symbiosis School of Biological Sciences, Symbiosis International (Deemed University), Pune, India
| | - Vaijayanti Kale
- Symbiosis Centre for Stem Cell Research, Symbiosis International (Deemed University), Pune, India
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11
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Meng L, Wei Y, Liang Y, Hu Q, Xie H. Stem cell homing in periodontal tissue regeneration. Front Bioeng Biotechnol 2022; 10:1017613. [PMID: 36312531 PMCID: PMC9607953 DOI: 10.3389/fbioe.2022.1017613] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2022] [Accepted: 10/03/2022] [Indexed: 11/29/2022] Open
Abstract
The destruction of periodontal tissue is a crucial problem faced by oral diseases, such as periodontitis and tooth avulsion. However, regenerating periodontal tissue is a huge clinical challenge because of the structural complexity and the poor self-healing capability of periodontal tissue. Tissue engineering has led to advances in periodontal regeneration, however, the source of exogenous seed cells is still a major obstacle. With the improvement of in situ tissue engineering and the exploration of stem cell niches, the homing of endogenous stem cells may bring promising treatment strategies in the future. In recent years, the applications of endogenous cell homing have been widely reported in clinical tissue repair, periodontal regeneration, and cell therapy prospects. Stimulating strategies have also been widely studied, such as the combination of cytokines and chemokines, and the implantation of tissue-engineered scaffolds. In the future, more research needs to be done to improve the efficiency of endogenous cell homing and expand the range of clinical applications.
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Affiliation(s)
- Lingxi Meng
- State Key Laboratory of Oral Diseases, Department of Head and Neck Oncology Surgery, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Yige Wei
- State Key Laboratory of Oral Diseases, Department of Head and Neck Oncology Surgery, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Yaxian Liang
- State Key Laboratory of Oral Diseases, Department of Head and Neck Oncology Surgery, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Qin Hu
- Faculty of Dentistry, The University of Hong Kong, Hong Kong, China
| | - Huixu Xie
- State Key Laboratory of Oral Diseases, Department of Head and Neck Oncology Surgery, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
- *Correspondence: Huixu Xie,
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12
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Khoshnood N, Shahrezaee MH, Shahrezaee M, Zamanian A. Three‐dimensional
bioprinting of tragacanth/hydroxyapaptite modified alginate bioinks for bone tissue engineering with tunable printability and bioactivity. J Appl Polym Sci 2022. [DOI: 10.1002/app.52833] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Affiliation(s)
- Negin Khoshnood
- Biomaterials Research Group Nanotechnology and Advanced Materials Department, Materials and Energy Research Center (MERC) Tehran Iran
| | | | - Mostafa Shahrezaee
- Department of Orthopedic Surgery, School of Medicine AJA University of Medical Science Tehran Iran
| | - Ali Zamanian
- Biomaterials Research Group Nanotechnology and Advanced Materials Department, Materials and Energy Research Center (MERC) Tehran Iran
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13
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Tevlin R, desJardins-Park H, Huber J, DiIorio S, Longaker M, Wan D. Musculoskeletal tissue engineering: Adipose derived stromal cell implementation for the treatment of osteoarthritis. Biomaterials 2022; 286:121544. [DOI: 10.1016/j.biomaterials.2022.121544] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Revised: 06/23/2021] [Accepted: 09/13/2021] [Indexed: 11/02/2022]
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14
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Li J, Zhang Y, Zhou X, Wang S, Hao R, Han J, Li M, Zhao Y, Chen C, Xu H. Enzymatically functionalized RGD-gelatin scaffolds that recruit host mesenchymal stem cells in vivo and promote bone regeneration. J Colloid Interface Sci 2022; 612:377-391. [PMID: 34998197 DOI: 10.1016/j.jcis.2021.12.091] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2021] [Revised: 11/20/2021] [Accepted: 12/14/2021] [Indexed: 01/27/2023]
Abstract
Critical-size bone defects are imposing a substantial biomedical burden. Despite being long regarded as a potential approach to mitigate this burden or an alternative to bone grafts, bone tissue engineering (BTE) has virtually not proceeded to widespread clinical practices. In the BTE field, it is highly required to find a facile method to prepare active scaffolds with tailored biological functions. Here, we immobilized cell adhesive RGD motifs onto gelatin sponge (GS) scaffolds through enzymatic linking. On the basis of the resulting RGD-functionalized GS (RGD/GS) scaffolds, we developed a new and convenient strategy for bone defect repair, in which the scaffolds were first used to recruit mesenchymal stem cells (MSCs) from skeletal muscle, immediately followed by their engraftment into bone defect. We demonstrated significantly enhanced host cells homing into RGD/GS scaffolds as a result of specific RGD-integrin interactions, and the recruited host cells showed a strong osteogenic differentiation potential. After ectopic implantation of cell-laden RGD/GS scaffolds into critical-size mouse bone defects, marked bone tissue regeneration occurred. The presented strategy not only provides an agile route for the preparation of bioactive scaffolds and the construction of osteoinductive bone-graft substitutes, but also avoids or minimizes the complicated and laborious cell isolation, in vitro expansion and cell seeding procedures used in the conventional BTE.
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Affiliation(s)
- Junling Li
- Department of Biological and Energy Chemical Engineering, College of Chemical Engineering, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China; Biomedical Sciences College & Shandong Medicinal Biotechnology Centre, Key Lab for Biotech-Drugs of National Health Commission, Shandong First Medical University & Shandong Academy of Medical Sciences, 6699 Qingdao Road, Ji'nan 250117, China
| | - Yan Zhang
- Department of Biological and Energy Chemical Engineering, College of Chemical Engineering, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China
| | - Xing Zhou
- Qingdao West Coast New Area Marine Development Bureau, 59 Shuilingshan Road, Qingdao 266400, China
| | - Shili Wang
- Biomedical Sciences College & Shandong Medicinal Biotechnology Centre, Key Lab for Biotech-Drugs of National Health Commission, Shandong First Medical University & Shandong Academy of Medical Sciences, 6699 Qingdao Road, Ji'nan 250117, China
| | - Ruirui Hao
- Department of Biological and Energy Chemical Engineering, College of Chemical Engineering, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China
| | - Jinxiang Han
- Biomedical Sciences College & Shandong Medicinal Biotechnology Centre, Key Lab for Biotech-Drugs of National Health Commission, Shandong First Medical University & Shandong Academy of Medical Sciences, 6699 Qingdao Road, Ji'nan 250117, China
| | - Mian Li
- Biomedical Sciences College & Shandong Medicinal Biotechnology Centre, Key Lab for Biotech-Drugs of National Health Commission, Shandong First Medical University & Shandong Academy of Medical Sciences, 6699 Qingdao Road, Ji'nan 250117, China
| | - Yurong Zhao
- Department of Biological and Energy Chemical Engineering, College of Chemical Engineering, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China
| | - Cuixia Chen
- Department of Biological and Energy Chemical Engineering, College of Chemical Engineering, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China.
| | - Hai Xu
- Department of Biological and Energy Chemical Engineering, College of Chemical Engineering, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China.
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15
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Zimmermann CE, Mackens-Kiani L, Acil Y, Terheyden H. Characterization of porcine mesenchymal stromal cells and their proliferative and osteogenic potential in long-term culture. J Stem Cells Regen Med 2022; 17:49-55. [PMID: 35250201 DOI: 10.46582/jsrm.1702008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Accepted: 10/07/2021] [Indexed: 12/29/2022]
Abstract
Background: Porcine mesenchymal stromal cells (pMSCs) are considered a valuable research model for bone tissue engineering, which requires adequate amounts of viable cells with sufficient potential for osteogenic differentiation. For isolation and expansion of these cells through long-term culture, appropriate culture conditions are needed. Objective: To study the effect of extended in vitro cultivation on pMSC proliferation and differentiation potential using different osteogenic and adipogenic induction media. Methods: pMSCs were isolated from the bone marrow of adult Göttingen minipigs, cultured, expanded to passage 20 (~160 days) and characterized by their expression of cell surface markers (wCD44, CD45, CD90, SWC9, fibronectin), alkaline phosphatase (ALP), and osteocalcin and their potential for osteogenic and adipogenic differentiation using different induction media. Results: pMSCs retained their capacity for proliferation and osteogenic differentiation, and the number of CD90-positive cells increased significantly over more than 60 population doublings. CD90 expression in uninduced cells correlated strongly with ALP expression following osteogenic induction. Medium enriched with calcium yielded a stronger osteogenic response. Conclusion: The selection of CD90-positive MSCs and adequate levels of calcium seem to enhance the osteogenic phenotype for bone tissue engineering.
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Affiliation(s)
- Corinna E Zimmermann
- Department of Craniomaxillofacial Surgery, University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller-Strasse 3, 24105 Kiel, Germany.,University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany
| | | | - Yahya Acil
- Department of Craniomaxillofacial Surgery, University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller-Strasse 3, 24105 Kiel, Germany
| | - Hendrik Terheyden
- Department of Craniomaxillofacial Surgery, University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller-Strasse 3, 24105 Kiel, Germany
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16
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Ohori-Morita Y, Niibe K, Limraksasin P, Nattasit P, Miao X, Yamada M, Mabuchi Y, Matsuzaki Y, Egusa H. OUP accepted manuscript. Stem Cells Transl Med 2022; 11:434-449. [PMID: 35267026 PMCID: PMC9052431 DOI: 10.1093/stcltm/szab030] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Accepted: 12/02/2021] [Indexed: 11/14/2022] Open
Affiliation(s)
- Yumi Ohori-Morita
- Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
| | - Kunimichi Niibe
- Corresponding authors: Kunimichi Niibe, DDS, PhD, Associate Professor, Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai-city, Miyagi 980-8575, Japan. Tel: +81-22-717-8363; Fax: +81-22-717-8367;
| | - Phoonsuk Limraksasin
- Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
| | - Praphawi Nattasit
- Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
| | - Xinchao Miao
- Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
| | - Masahiro Yamada
- Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
| | - Yo Mabuchi
- Department of Biochemistry and Biophysics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Yumi Matsuzaki
- Department of Life Science, Faculty of Medicine, Shimane University, 89-1 Enya-cho, Izumo, Shimane 693-8501, Japan
| | - Hiroshi Egusa
- Hiroshi Egusa, DDS, PhD, Director, Center for Advanced Stem Cell and Regenerative Research, Professor and Chair, Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai-city 980-8575, Japan. Tel: +81-22-717-8363; Fax: +81-22-717-8367;
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17
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Chondrogenic Characteristics of Auricular Chondrocytes Cocultured With Adipose-Derived Stem Cells are Superior to Stromal Vascular Fraction of Adipose Tissue. J Craniofac Surg 2021; 32:2906-2911. [PMID: 34727488 DOI: 10.1097/scs.0000000000007902] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
Abstract
ABSTRACT Reconstruction of craniofacial cartilage defects is among the most challenging operations in facial plastic surgery. The co-culture system of partial replacement of chondrocytes by stem cells has been confirmed effective in the repair of cartilaginous defects. The aim of this study is to compare chondrogenic properties of expanded adipose-derived stem cells (ADSCs) and stromal vascular fraction (SVF), including ADSCs/SVF monoculture and coculture with rabbit auricular chondrocytes (ACs). Analysis of morphology, histology, real-time polymerase chain reaction and glycosaminoglycans (GAG) quantification were performed to characterize the chondrogenesis of pellets. The triple differentiation potential of ADSCs had been confirmed. Further, using flow cytometry, the authors demonstrated that ADSCs and SVF have different characteristics in cell surface markers, and ADSCs are more enriched in cells from the mesenchymal lineage than SVF. GAG production of ADSCs is significantly higher than that of SVF in pellet monoculture, and pellet coculture of ADSCs and ACs are better in depositing cartilage matrix than the mixture of SVF and ACs. Our study suggests that ADSCs may be more suitable seed cells for craniofacial cartilage defect or deformity repair.
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18
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Application of Human Adipose-Derived Stem cells for Bone Regeneration of the Skull in Humans. J Craniofac Surg 2021; 33:360-363. [PMID: 34636755 DOI: 10.1097/scs.0000000000008114] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
Abstract
BACKGROUND Archeological archives report cranioplasty as 1 of the oldest surgical procedures; however, it was not until the last century that true advances have been made. Alternative approaches are necessary to achieve optimal closure of the defect with fewer adverse effects. We aim to evaluate the use of human adipose-derived stem cells (hADSCs) alone or seeded in scaffolds as the main treatment for cranial bone defects and to assess human patient outcomes. METHODS A systematic review was performed by querying PubMed, Ovid MEDLINE, EMBASE, and Cumulative Index to Nursing and Allied Health Literature databases with the MeSH terms: "adipose-derived stem cells," "cranial bone defect," "stromal vascular factor," "fat grafting," as well as synonyms in combinations determined by our search strategy. We included human models that used hADSCs as primary therapy. We excluded studies in languages other than English. RESULTS One hundred ninety-four studies were identified after removal of duplicates. Four articles that used hADSCs as the main therapy to treat calvarial defects in humans were included. One article applied the cell therapy alone, and 3 used β-tricalcium phosphate granules as a scaffold to seed the hADSCs. CONCLUSIONS Bone regeneration was reached in a short and intermediate period using autologous hADSCs in humans with no major adverse effects in all 4 articles included. A long-term follow-up study (6 years) exhibited late infections and reabsorption of the β-tricalcium phosphate scaffold seeded with hADSCs.
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19
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Le Q, Madhu V, Hart JM, Farber CR, Zunder ER, Dighe AS, Cui Q. Current evidence on potential of adipose derived stem cells to enhance bone regeneration and future projection. World J Stem Cells 2021; 13:1248-1277. [PMID: 34630861 PMCID: PMC8474721 DOI: 10.4252/wjsc.v13.i9.1248] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/28/2021] [Revised: 05/22/2021] [Accepted: 08/18/2021] [Indexed: 02/06/2023] Open
Abstract
Injuries to the postnatal skeleton are naturally repaired through successive steps involving specific cell types in a process collectively termed “bone regeneration”. Although complex, bone regeneration occurs through a series of well-orchestrated stages wherein endogenous bone stem cells play a central role. In most situations, bone regeneration is successful; however, there are instances when it fails and creates non-healing injuries or fracture nonunion requiring surgical or therapeutic interventions. Transplantation of adult or mesenchymal stem cells (MSCs) defined by the International Society for Cell and Gene Therapy (ISCT) as CD105+CD90+CD73+CD45-CD34-CD14orCD11b-CD79αorCD19-HLA-DR- is being investigated as an attractive therapy for bone regeneration throughout the world. MSCs isolated from adipose tissue, adipose-derived stem cells (ADSCs), are gaining increasing attention since this is the most abundant source of adult stem cells and the isolation process for ADSCs is straightforward. Currently, there is not a single Food and Drug Administration (FDA) approved ADSCs product for bone regeneration. Although the safety of ADSCs is established from their usage in numerous clinical trials, the bone-forming potential of ADSCs and MSCs, in general, is highly controversial. Growing evidence suggests that the ISCT defined phenotype may not represent bona fide osteoprogenitors. Transplantation of both ADSCs and the CD105- sub-population of ADSCs has been reported to induce bone regeneration. Most notably, cells expressing other markers such as CD146, AlphaV, CD200, PDPN, CD164, CXCR4, and PDGFRα have been shown to represent osteogenic sub-population within ADSCs. Amongst other strategies to improve the bone-forming ability of ADSCs, modulation of VEGF, TGF-β1 and BMP signaling pathways of ADSCs has shown promising results. The U.S. FDA reveals that 73% of Investigational New Drug applications for stem cell-based products rely on CD105 expression as the “positive” marker for adult stem cells. A concerted effort involving the scientific community, clinicians, industries, and regulatory bodies to redefine ADSCs using powerful selection markers and strategies to modulate signaling pathways of ADSCs will speed up the therapeutic use of ADSCs for bone regeneration.
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Affiliation(s)
- Quang Le
- Department of Orthopaedic Surgery, University of Virginia School of Medicine, Charlottesville, VA 22908, United States
| | - Vedavathi Madhu
- Orthopaedic Surgery Research, Thomas Jefferson University, Philadelphia, PA 19107, United States
| | - Joseph M Hart
- Department of Orthopaedic Surgery, University of Virginia School of Medicine, Charlottesville, VA 22908, United States
| | - Charles R Farber
- Center for Public Health Genomics, University of Virginia, Charlottesville, VA 22908, United States
- Departments of Public Health Sciences and Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, United States
| | - Eli R Zunder
- Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, United States
| | - Abhijit S Dighe
- Department of Orthopaedic Surgery, University of Virginia School of Medicine, Charlottesville, VA 22908, United States
| | - Quanjun Cui
- Department of Orthopaedic Surgery, University of Virginia School of Medicine, Charlottesville, VA 22908, United States
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20
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The Gingiva from the Tissue Surrounding the Bone to the Tissue Regenerating the Bone: A Systematic Review of the Osteogenic Capacity of Gingival Mesenchymal Stem Cells in Preclinical Studies. Stem Cells Int 2021; 2021:6698100. [PMID: 34234830 PMCID: PMC8218920 DOI: 10.1155/2021/6698100] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2020] [Revised: 12/20/2020] [Accepted: 05/21/2021] [Indexed: 12/13/2022] Open
Abstract
The current review aims to systematically assess the osteogenic capacity of gingiva-derived mesenchymal stem cells (GMSCs) in preclinical studies. A comprehensive electronic search of PubMed, Embase, Web of Science, and Scopus databases, as well as a manual search of relevant references, was performed in June 2020 without date or language restrictions. Eligibility criteria were the following: studies that compared mesenchymal stem cells (MSCs) derived from the gingiva with other MSC sources (in vitro or in vivo) or cell-free scaffold (in vivo) and studies that reported at least one of the following outcomes: osteogenic potential and new bone formation for in vitro and in vivo, respectively. Moreover, the assessment of included studies was conducted using appropriate guidelines. From 646 initial retrieved studies, 35 full-text articles were subjected to further screening and 26 studies were selected (20 in vitro studies and 6 in vivo studies). GMSCs showed great proliferation capacity and expressed recognized mesenchymal stem cell markers, particularly CD90. In vitro, MSC sources including GMSCs were capable of undergoing osteogenic differentiation with less ability in GMSCs, while most in vivo studies confirmed the capacity of GMSCs to regenerate bony defects. Concerning the assessment of methodological quality, in vitro studies met the relevant guideline except in five areas: the sample size calculation, randomization, allocation concealment, implementation, and blinding, and in vivo publications had probably low risk of bias in most domains except in three areas: allocation concealment, attrition, and blinding items.
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21
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Characterization of encapsulated porcine cardiosphere-derived cells embedded in 3D alginate matrices. Int J Pharm 2021; 599:120454. [PMID: 33676988 DOI: 10.1016/j.ijpharm.2021.120454] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2021] [Revised: 02/23/2021] [Accepted: 03/01/2021] [Indexed: 12/22/2022]
Abstract
Myocardial infarction is caused by an interruption of coronary blood flow, leading to one of the main death causes worldwide. Current therapeutic approaches are palliative and not able to solve the loss of cardiac tissue. Cardiosphere derived cells (CDCs) reduce scarring, and increase viable myocardium, with safety and adequate biodistribution, but show a low rate engraftment and survival after implantation. In order to solve the low retention, we propose the encapsulation of CDCs within three-dimensional alginate-poly-L-lysine-alginate matrix as therapy for cardiac regeneration. In this work, we demonstrate the encapsulation of CDCs in alginate matrix, with no decrease in viability over a month, and showing the preservation of CDCs phenotype, differentiation potential, gene expression profile and growth factor release after encapsulation, moving a step forward to clinical translation of CDCs therapy in regeneration in heart failure.
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22
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Multiplex Analysis of Adipose-Derived Stem Cell (ASC) Immunophenotype Adaption to In Vitro Expansion. Cells 2021; 10:cells10020218. [PMID: 33499095 PMCID: PMC7911224 DOI: 10.3390/cells10020218] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2020] [Revised: 01/11/2021] [Accepted: 01/19/2021] [Indexed: 12/15/2022] Open
Abstract
In order to enhance the therapeutic potential, it is important that sufficient knowledge regarding the dynamic changes of adipose-derived stem cell (ASC) immunophenotypical and biological properties during in vitro growth is available. Consequently, we embarked on a study to follow the evolution of highly defined cell subsets from three unrelated donors in the course of eight passages on tissue culture polystyrene. The co-expression patterns were defined by panels encompassing seven and five cell surface markers, including CD34, CD146, CD166, CD200, CD248, CD271, and CD274 and CD29, CD31, CD36, CD201, and Stro-1, respectively. The analysis was performed using multichromatic flow cytometry. We observed a major paradigm shift, where the CD166-CD34+ combination which was found across all cell subsets early in the culture was replaced by the CD166+ phenotype as the population homogeneity increased with time. At all analysis points, the cultures were dominated by a few major clones that were highly prevalent in most of the donors. The selection process resulted in two predominant clones in the larger panel (CD166+CD34-CD146-CD271- CD274-CD248-CD200- and CD166+CD34+ CD146-CD271-CD274-CD248-CD200-) and one clone in the smaller panel (CD29+CD201+CD36- Stro-1- CD31-). The minor subsets, including CD166+CD34-CD146-CD271+CD274-CD248-CD200- and CD166+CD34+CD146+CD271-CD274-CD248-CD200-, and CD29+CD201-CD36-Stro-1-CD31-, CD29+CD201+CD36-Stro-1+CD31-, and CD29+CD201+CD36+Stro-1-CD31-, in the seven and five marker panels, respectively, were, on the other, hand highly fluctuating and donor-dependent. The results demonstrate that only a limited number of phenotypical repertoires are possible in ASC cultures. Marked differences in their relative occurrence between distinct individuals underscore the need for potency standardization of different ASC preparation to improve the clinical outcome.
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23
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Lee EJ, Jain M, Alimperti S. Bone Microvasculature: Stimulus for Tissue Function and Regeneration. TISSUE ENGINEERING PART B-REVIEWS 2020; 27:313-329. [PMID: 32940150 DOI: 10.1089/ten.teb.2020.0154] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Bone is a highly vascularized organ, providing structural support to the body, and its development, regeneration, and remodeling depend on the microvascular homeostasis. Loss or impairment of vascular function can develop diseases, such as large bone defects, avascular necrosis, osteoporosis, osteoarthritis, and osteopetrosis. In this review, we summarize how vasculature controls bone development and homeostasis in normal and disease cases. A better understanding of this process will facilitate the development of novel disease treatments that promote bone regeneration and remodeling. Specifically, approaches based on tissue engineering components, such as stem cells and growth factors, have demonstrated the capacity to induce bone microvasculature regeneration and mineralization. This knowledge will have relevant clinical implications for the treatment of bone disorders by developing novel pharmaceutical approaches and bone grafts. Finally, the tissue engineering approaches incorporating vascular components may widely be applied to treat other organ diseases by enhancing their regeneration capacity. Impact statement Bone vasculature is imperative in the process of bone development, regeneration, and remodeling. Alterations or disruption of the bone vasculature leads to loss of bone homeostasis and the development of bone diseases. In this study, we review the role of vasculature on bone diseases and how vascular tissue engineering strategies, with a detailed emphasis on the role of stem cells and growth factors, will contribute to bone therapeutics.
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Affiliation(s)
- Eun-Jin Lee
- American Dental Association Science and Research Institute, Gaithersburg, Maryland, USA
| | - Mahim Jain
- Kennedy Krieger Institute, John Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Stella Alimperti
- American Dental Association Science and Research Institute, Gaithersburg, Maryland, USA
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24
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Kannan S, Ghosh J, Dhara SK. Osteogenic differentiation potential of porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media. Biol Open 2020; 9:bio053280. [PMID: 32973080 PMCID: PMC7595700 DOI: 10.1242/bio.053280] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2020] [Accepted: 09/09/2020] [Indexed: 12/25/2022] Open
Abstract
Multipotent porcine mesenchymal stem cells (pMSC) are invaluable for research and therapeutic use in regenerative medicine. Media used for derivation and expansion of pMSC may play an important role for the selection of MSC subpopulation at an early stage and thereby, the specific basal medium may also affect differentiation potential of these cells. The present study was undertaken to evaluate the effects of αMEM, aDMEM, M199, αMEM/M199, aDMEM/M199 and αMEM/aDMEM media on (1) porcine bone marrow MSC derivation; (2) expression of number of osteogenic markers (ALP, COL1A1, SPP1 and BGLAP) at 5th and 10th passage in pMSC before differentiation; and (3) differentiation of pMSC (at 5th passage) to osteogenic lineage. Morphological changes and matrix formation in osteogenic cells were evaluated by microscopic examination. Calcium deposits in osteocytes were confirmed by Alizarin Red S staining. Based on expression of different markers, it was evident that selection of bone marrow pMSC subpopulations was independent of basal media used. However, the differentiation of those pMSCs, specifically to osteogenic lineage, was dependent on the medium used for expansion of pMSC at the pre-differentiation stage. We demonstrated here that the pMSC grown in combined αMEM/aDMEM (1:1) medium expressed number of osteogenic markers and these pMSC underwent osteogenic differentiation most efficiently, in comparison to porcine mesenchymal stem cells grown in other media. In conclusion, osteogenic differentiation potential of pMSC maintained in αMEM/aDMEM medium was observed significantly higher compared to cells cultivated in other media and therefore, the combined medium αMEM/aDMEM (1:1) may preferentially be used for expansion of pMSC, if needed for osteogenic differentiation.
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Affiliation(s)
- Sangeetha Kannan
- Department of Biotechnology, Jain University, Bangalore 560011, Karnataka, India
| | - Jyotirmoy Ghosh
- Molecular Biology Laboratory, ICAR-National Institute of Animal Nutrition and Physiology, Bangalore 560030, Karnataka, India
| | - Sujoy K Dhara
- Stem Cell Laboratory, Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243122, India
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25
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Palombella S, Guiotto M, Higgins GC, Applegate LL, Raffoul W, Cherubino M, Hart A, Riehle MO, di Summa PG. Human platelet lysate as a potential clinical-translatable supplement to support the neurotrophic properties of human adipose-derived stem cells. Stem Cell Res Ther 2020; 11:432. [PMID: 33023632 PMCID: PMC7537973 DOI: 10.1186/s13287-020-01949-4] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2020] [Accepted: 09/24/2020] [Indexed: 02/06/2023] Open
Abstract
Background The autologous nerve graft, despite its donor site morbidity and unpredictable functional recovery, continues to be the gold standard in peripheral nerve repair. Rodent research studies have shown promising results with cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit, as an alternative strategy for nerve regeneration. To achieve meaningful clinical translation, cell therapy must comply with biosafety. Cell extraction and expansion methods that use animal-derived products, including enzymatic adipose tissue dissociation and the use of fetal bovine serum (FBS) as a culture medium supplement, have the potential for transmission of zoonotic infectious and immunogenicity. Human-platelet-lysate (hPL) serum has been used in recent years in human cell expansion, showing reliability in clinical applications. Methods We investigated whether hADSC can be routinely isolated and cultured in a completely xenogeneic-free way (using hPL culture medium supplement and avoiding collagenase digestion) without altering their physiology and stem properties. Outcomes in terms of stem marker expression (CD105, CD90, CD73) and the osteocyte/adipocyte differentiation capacity were compared with classical collagenase digestion and FBS-supplemented hADSC expansion. Results We found no significant differences between the two examined extraction and culture protocols in terms of cluster differentiation (CD) marker expression and stem cell plasticity, while hADSC in hPL showed a significantly higher proliferation rate when compared with the usual FBS-added medium. Considering the important key growth factors (particularly brain-derived growth factor (BDNF)) present in hPL, we investigated a possible neurogenic commitment of hADSC when cultured with hPL. Interestingly, hADSC cultured in hPL showed a statistically higher secretion of neurotrophic factors BDNF, glial cell-derived growth factor (GDNF), and nerve-derived growth factor (NFG) than FBS-cultured cells. When cocultured in the presence of primary neurons, hADSC which had been grown under hPL supplementation, showed significantly enhanced neurotrophic properties. Conclusions The hPL-supplement medium could improve cell proliferation and neurotropism while maintaining stable cell properties, showing effectiveness in clinical translation and significant potential in peripheral nerve research.
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Affiliation(s)
- Silvia Palombella
- Unit of Regenerative Therapy, Service of Plastic, Reconstructive and Hand Surgery, Department of Musculoskeletal Medicine, Lausanne University Hospital, Lausanne, Switzerland. .,Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy.
| | - Martino Guiotto
- Department of Plastic, Reconstructive and Hand Surgery, Centre Hospitalier Universitaire Vaudois (CHUV), Rue du Bugnon, 21, 1011, Lausanne, Switzerland.,Centre for Cellular Microenvironment (CeMi), University of Glasgow, Glasgow, UK
| | - Gillian C Higgins
- Centre for Cellular Microenvironment (CeMi), University of Glasgow, Glasgow, UK.,Canniesburn Plastic Surgery Unit, Glasgow Royal Infirmary, Glasgow, UK
| | - Laurent L Applegate
- Unit of Regenerative Therapy, Service of Plastic, Reconstructive and Hand Surgery, Department of Musculoskeletal Medicine, Lausanne University Hospital, Lausanne, Switzerland
| | - Wassim Raffoul
- Department of Plastic, Reconstructive and Hand Surgery, Centre Hospitalier Universitaire Vaudois (CHUV), Rue du Bugnon, 21, 1011, Lausanne, Switzerland
| | - Mario Cherubino
- Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy
| | - Andrew Hart
- Centre for Cellular Microenvironment (CeMi), University of Glasgow, Glasgow, UK.,Canniesburn Plastic Surgery Unit, Glasgow Royal Infirmary, Glasgow, UK
| | - Mathis O Riehle
- Centre for Cellular Microenvironment (CeMi), University of Glasgow, Glasgow, UK
| | - Pietro G di Summa
- Department of Plastic, Reconstructive and Hand Surgery, Centre Hospitalier Universitaire Vaudois (CHUV), Rue du Bugnon, 21, 1011, Lausanne, Switzerland.
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26
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Wang Y, Negri S, Li Z, Xu J, Hsu CY, Peault B, Broderick K, James AW. Anti-DKK1 Enhances the Early Osteogenic Differentiation of Human Adipose-Derived Stem/Stromal Cells. Stem Cells Dev 2020; 29:1007-1015. [PMID: 32460636 DOI: 10.1089/scd.2020.0070] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Adipose-derived stem/stromal cells (ASCs) have been previously used for bone repair. However, significant cell heterogeneity exists within the ASC population, which has the potential to result in unreliable bone tissue formation and/or low efficacy. Although the use of cell sorting to lower cell heterogeneity is one method to improve bone formation, this is a technically sophisticated and costly process. In this study, we tried to find a simpler and more deployable solution-blocking antiosteogenic molecule Dickkopf-1 (DKK1) to improve osteogenic differentiation. Human adipose-derived stem cells were derived from = 5 samples of human lipoaspirate. In vitro, anti-DKK1 treatment, but not anti-sclerostin (SOST), promoted ASC osteogenic differentiation, assessed by alizarin red staining and real-time polymerase chain reaction (qPCR). Increased canonical Wnt signaling was confirmed after anti-DKK1 treatment. Expression levels of DKK1 peaked during early osteogenic differentiation (day 3). Concordantly, anti-DKK1 supplemented early (day 3 or before), but not later (day 7) during osteogenic differentiation positively regulated osteoblast formation. Finally, anti-DKK1 led to increased transcript abundance of the Wnt inhibitor SOST, potentially representing a compensatory cellular mechanism. In sum, DKK1 represents a targetable "molecular brake" on the osteogenic differentiation of human ASC. Moreover, release of this brake by neutralizing anti-DKK1 antibody treatment at least partially rescues the poor bone-forming efficacy of ASC.
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Affiliation(s)
- Yiyun Wang
- Department of Pathology and Johns Hopkins University, Baltimore, Maryland, USA
| | - Stefano Negri
- Department of Pathology and Johns Hopkins University, Baltimore, Maryland, USA
| | - Zhao Li
- Department of Pathology and Johns Hopkins University, Baltimore, Maryland, USA
| | - Jiajia Xu
- Department of Pathology and Johns Hopkins University, Baltimore, Maryland, USA
| | - Ching-Yun Hsu
- Department of Pathology and Johns Hopkins University, Baltimore, Maryland, USA
| | - Bruno Peault
- UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, Pittsburgh, Pennsylvania, USA.,Center for Cardiovascular Science and MRC Center for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
| | - Kristen Broderick
- Department of Plastic Surgery, Johns Hopkins University, Baltimore, Maryland, USA
| | - Aaron W James
- Department of Pathology and Johns Hopkins University, Baltimore, Maryland, USA.,UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, Pittsburgh, Pennsylvania, USA
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27
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Borrelli MR, Patel RA, Adem S, Diaz Deleon NM, Shen AH, Sokol J, Yen S, Chang EY, Nazerali R, Nguyen D, Momeni A, Wang KC, Longaker MT, Wan DC. The antifibrotic adipose-derived stromal cell: Grafted fat enriched with CD74+ adipose-derived stromal cells reduces chronic radiation-induced skin fibrosis. Stem Cells Transl Med 2020; 9:1401-1413. [PMID: 32563212 PMCID: PMC7581454 DOI: 10.1002/sctm.19-0317] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2019] [Revised: 03/04/2020] [Accepted: 03/27/2020] [Indexed: 02/06/2023] Open
Abstract
Fat grafting can reduce radiation‐induced fibrosis. Improved outcomes are found when fat grafts are enriched with adipose‐derived stromal cells (ASCs), implicating ASCs as key drivers of soft tissue regeneration. We have identified a subpopulation of ASCs positive for CD74 with enhanced antifibrotic effects. Compared to CD74− and unsorted (US) ASCs, CD74+ ASCs have increased expression of hepatocyte growth factor, fibroblast growth factor 2, and transforming growth factor β3 (TGF‐β3) and decreased levels of TGF‐β1. Dermal fibroblasts incubated with conditioned media from CD74+ ASCs produced less collagen upon stimulation, compared to fibroblasts incubated with media from CD74− or US ASCs. Upon transplantation, fat grafts enriched with CD74+ ASCs reduced the stiffness, dermal thickness, and collagen content of overlying skin, and decreased the relative proportions of more fibrotic dermal fibroblasts. Improvements in several extracellular matrix components were also appreciated on immunofluorescent staining. Together these findings indicate CD74+ ASCs have antifibrotic qualities and may play an important role in future strategies to address fibrotic remodeling following radiation‐induced fibrosis.
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Affiliation(s)
- Mimi R Borrelli
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Ronak A Patel
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Sandeep Adem
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Nestor M Diaz Deleon
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Abra H Shen
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Jan Sokol
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Sara Yen
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Erin Y Chang
- Program in Epithelial Biology, Department of Dermatology, Stanford University School of Medicine, Stanford, California, USA
| | - Rahim Nazerali
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Dung Nguyen
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Arash Momeni
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Kevin C Wang
- Program in Epithelial Biology, Department of Dermatology, Stanford University School of Medicine, Stanford, California, USA
| | - Michael T Longaker
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA.,Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA
| | - Derrick C Wan
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
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Borrelli MR, Patel RA, Blackshear C, Vistnes S, Diaz Deleon NM, Adem S, Shen AH, Sokol J, Momeni A, Nguyen D, Longaker MT, Wan DC. CD34+CD146+ adipose-derived stromal cells enhance engraftment of transplanted fat. Stem Cells Transl Med 2020; 9:1389-1400. [PMID: 32543083 PMCID: PMC7581443 DOI: 10.1002/sctm.19-0195] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2019] [Revised: 04/24/2020] [Accepted: 05/24/2020] [Indexed: 12/16/2022] Open
Abstract
Fat grafting is a surgical technique able to reconstruct and regenerate soft tissue. The adipose‐derived stromal cells (ASCs) within the stromal vascular fraction are believed to drive these beneficial effects. ASCs are increasingly recognized to be a heterogeneous group, comprised of multiple stem and progenitor subpopulations with distinct functions. We hypothesized the existence of an ASC subpopulation with enhanced angiogenic potential. Human ASCs that were CD34+CD146+, CD34+CD146−, or CD34+ unfractionated (UF) were isolated by flow cytometry for comparison of expression of proangiogenic factors and endothelial tube‐forming potential. Next, lipoaspirate was enriched with either CD34+CD146+, CD34+CD146−, CD34+ UF ASCs, or was not enriched, and grafted beneath the scalp skin of immunodeficient CD‐1 Nude mice (10 000 cells/200 μL/graft). Fat retention was monitored radiographically more than 8 weeks and fat grafts were harvested for histological assessment of quality and vascularization. The CD34+CD146+ subpopulation comprised ~30% of ASCs, and exhibited increased expression of vascular endothelial growth factor and angiopoietin‐1 compared to CD34+CD146− and CD34+ UF ASCs, and increased expression of fibroblast growth factor‐2 compared to CD34+CD146− ASCs. The CD34+CD146+ subpopulation exhibited enhanced induction of tube‐formation compared to CD34+CD146− ASCs. Upon transplantation, fat enriched CD34+CD146+ ASCs underwent less resorption and had improved histologic quality and vascularization. We have identified a subpopulation of CD34+ ASCs with enhanced angiogenic effects in vitro and in vivo, likely mediated by increased expression of potent proangiogenic factors. These findings suggest that enriching lipoaspirate with CD34+CD146+ ASCs may enhance fat graft vascularization and retention in the clinical setting.
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Affiliation(s)
- Mimi R Borrelli
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Ronak A Patel
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Charles Blackshear
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Stephanie Vistnes
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Nestor M Diaz Deleon
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Sandeep Adem
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Abra H Shen
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Jan Sokol
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Arash Momeni
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Dung Nguyen
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Michael T Longaker
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA.,Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA
| | - Derrick C Wan
- Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic Surgery, Stanford University School of Medicine, Stanford, California, USA
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29
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Sangeetha KN, Vennila R, Secunda R, Sakthivel S, Pathak S, Jeswanth S, Surendran R. Functional variations between Mesenchymal Stem Cells of different tissue origins: A comparative gene expression profiling. Biotechnol Lett 2020; 42:1287-1304. [PMID: 32372268 DOI: 10.1007/s10529-020-02898-x] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2019] [Accepted: 04/24/2020] [Indexed: 12/26/2022]
Abstract
BACKGROUND Mesenchymal Stem Cells (MSCs), regardless of the tissue sources, are considered as excellent candidates for cellular therapy as they are immune-privileged cells containing a multitude of therapeutic functions that aid in tissue regeneration and repair. For the effective application of these cells in cell therapy, it is important to understand and characterize their biological functions. OBJECTIVES The present study attempts to characterize the variations in multipotent function such as cell surface antigen levels, proliferation, differentiation and stemness (pluripotency) potential of MSCs isolated from foetal [wharton's jelly (WJ), foetal and maternal side of placenta (PF and PM)] and adult tissue sources [bone marrow (BM) and adipose tissue (AT)] using gene expression by real time PCR (qRT-PCR). RESULTS Amongst the different tissue sources, PM, PF and AT-MSCs exhibited significant increase (p < 0.001, p < 0.001 and p < 0.01 respectively) in CD 73 expression and therefore could have a role in immunomodulation. WJ-MSCs exhibited superior proliferation potential based on growth curve, PCNA and Wnt gene expression. BM-MSCs were superior in exhibiting trilineage differentiation. Enhanced stemness potential (Oct 4 and Nanog) was observed for both BM and WJ-MSCs. In addition, BM and WJ-MSCs expressed high levels of CD 90 making them suitable in bone repair and regeneration. CONCLUSION Thus to conclude, out of the five different sources tested, BM an adult source and WJ-MSCs a foetal source were superior in exhibiting most of the biological functions indicating that these sources may be suitable candidates for cell repair and regeneration studies.
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Affiliation(s)
- K N Sangeetha
- Stem Cell Research Centre, Government Stanley Hospital, Chennai, Tamilnadu, 600001, India
| | | | - R Secunda
- Stem Cell Research Centre, Government Stanley Hospital, Chennai, Tamilnadu, 600001, India.
| | - S Sakthivel
- Stem Cell Research Centre, Government Stanley Hospital, Chennai, Tamilnadu, 600001, India
| | - Surajit Pathak
- Chettinad Academy of Research and Education, Chettinad Hospital & Research Institute, Chennai, Tamilnadu, India
| | - S Jeswanth
- Stem Cell Research Centre, Government Stanley Hospital, Chennai, Tamilnadu, 600001, India
| | - R Surendran
- Hepato-Pancreato-Biliary Centre for Surgery & Transplantation, MIOT International, Chennai, Tamilnadu, India
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Yang J, Zhan XZ, Malola J, Li ZY, Pawar JS, Zhang HT, Zha ZG. The multiple roles of Thy-1 in cell differentiation and regeneration. Differentiation 2020; 113:38-48. [PMID: 32403041 DOI: 10.1016/j.diff.2020.03.003] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2020] [Revised: 03/17/2020] [Accepted: 03/22/2020] [Indexed: 11/17/2022]
Abstract
Thy-1 is a 25-37 kDa glycophosphatidylinositol (GPI)-anchored cell surface protein that was discovered more than 50 years ago. Recent findings have suggested that Thy-1 is expressed on thymocytes, mesenchymal stem cells (MSCs), cancer stem cells, hematopoietic stem cells, fibroblasts, myofibroblasts, endothelial cells, neuronal smooth muscle cells, and pan T cells. Thy-1 plays vital roles in cell migration, adhesion, differentiation, transdifferentiation, apoptosis, mechanotransduction, and cell division, which in turn are involved in tumor development, pulmonary fibrosis, neurite outgrowth, and T cell activation. Studies have increasingly indicated a significant role of Thy-1 in cell differentiation and regeneration. However, despite recent research, many questions remain regarding the roles of Thy-1 in cell differentiation and regeneration. This review aimed to summarize the roles of Thy-1 in cell differentiation and regeneration. Furthermore, since Thy-1 is an outer leaflet membrane protein anchored by GPI, we attempted to address how Thy-1 regulates intracellular pathways through cis and trans signals. Due to the complexity and mystery surrounding the issue, we also summarized the Thy-1-related pathways in different biological processes, and this might provide novel insights in the field of cell differentiation and regeneration.
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Affiliation(s)
- Jie Yang
- Institute of Orthopedic Diseases and Department of Bone and Joint Surgery, the First Affiliated Hospital, Jinan University, Guangzhou, 510630, Guangdong, China
| | - Xiao-Zhen Zhan
- Department of Stomatology, the First Affiliated Hospital, Jinan University, Guangzhou, 510630, Guangdong, China
| | - Jonathan Malola
- College of Pharmacy, Purdue University, West Lafayette, 47906, IN, USA
| | - Zhen-Yan Li
- Institute of Orthopedic Diseases and Department of Bone and Joint Surgery, the First Affiliated Hospital, Jinan University, Guangzhou, 510630, Guangdong, China
| | - Jogendra Singh Pawar
- Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, 47906, IN, USA
| | - Huan-Tian Zhang
- Institute of Orthopedic Diseases and Department of Bone and Joint Surgery, the First Affiliated Hospital, Jinan University, Guangzhou, 510630, Guangdong, China.
| | - Zhen-Gang Zha
- Institute of Orthopedic Diseases and Department of Bone and Joint Surgery, the First Affiliated Hospital, Jinan University, Guangzhou, 510630, Guangdong, China.
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31
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Li Z, Mu D, Liu C, Xin M, Fu S, Li S, Qi J, Wang Q, Luan J. The cell yields and biological characteristics of stromal/stem cells from lipoaspirate with different digestion loading ratio. Cytotechnology 2020; 72:203-215. [PMID: 31993890 PMCID: PMC7193004 DOI: 10.1007/s10616-020-00369-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2019] [Accepted: 01/09/2020] [Indexed: 01/31/2023] Open
Abstract
Effective harvesting procedure for adipose tissue is demanded by the affordable Good Manufacturing Practice-Compliant Production of clinical-grade adipose tissue-derived stem cells (hADSCs). Enzymatic digestion using collagenase is the most reliable method of adipose tissue-derived stem cells (hADSCs) isolation, while the optimized loading volume ratios of digestion to container during the shaking process of adipose tissue and collagenase mixture are still lacking. This study was conducted to determine the optimized loading volume ratio (mixture to container) for enzymatic digestion of Stromal/Stem Cells from lipoaspirate. Lipoaspirates were obtained from twelve women immediately after liposuction. Then tissue from each patient was divided into four groups according to different loading volume ratios in 50 ml centrifugal tube: 0.2 group, 0.4 group, 0.6 group, 0.8 group. Stromal vascular fractions (SVF) were obtained from each group, then total cell counts, viability and viable cell count were performed. hADSCs were harvested at passage (P) 2, whose morphologies, immunophenotypes, proliferation, and tri-differentiation abilities were compared. 0.4 loading volume ratio provided the highest cell yield, favorable viability and viable cell yield. The proliferation and triple differentiation ability of hADSCs obtained by 0.4 group was not inferior to that of other groups. Therefore, 0.4 may be the optimal loading volume ratio for hADSCs isolation from lipoaspirate by enzymatic digestion in current setting.
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Affiliation(s)
- Zifei Li
- Breast Plastic and Reconstructive Surgery Center of Plastic Surgery Hospital, Chinese Academy of Medical Science and Peking Union Medical College, 33 Ba-Da-Chu Rd, Shijingshan Dist, Beijing, 100144, People's Republic of China
| | - Dali Mu
- Breast Plastic and Reconstructive Surgery Center of Plastic Surgery Hospital, Chinese Academy of Medical Science and Peking Union Medical College, 33 Ba-Da-Chu Rd, Shijingshan Dist, Beijing, 100144, People's Republic of China
| | - Chunjun Liu
- Breast Plastic and Reconstructive Surgery Center of Plastic Surgery Hospital, Chinese Academy of Medical Science and Peking Union Medical College, 33 Ba-Da-Chu Rd, Shijingshan Dist, Beijing, 100144, People's Republic of China
| | - Minqiang Xin
- Breast Plastic and Reconstructive Surgery Center of Plastic Surgery Hospital, Chinese Academy of Medical Science and Peking Union Medical College, 33 Ba-Da-Chu Rd, Shijingshan Dist, Beijing, 100144, People's Republic of China
| | - Su Fu
- Breast Plastic and Reconstructive Surgery Center of Plastic Surgery Hospital, Chinese Academy of Medical Science and Peking Union Medical College, 33 Ba-Da-Chu Rd, Shijingshan Dist, Beijing, 100144, People's Republic of China
| | - Shangshan Li
- Breast Plastic and Reconstructive Surgery Center of Plastic Surgery Hospital, Chinese Academy of Medical Science and Peking Union Medical College, 33 Ba-Da-Chu Rd, Shijingshan Dist, Beijing, 100144, People's Republic of China
| | - Jun Qi
- Breast Plastic and Reconstructive Surgery Center of Plastic Surgery Hospital, Chinese Academy of Medical Science and Peking Union Medical College, 33 Ba-Da-Chu Rd, Shijingshan Dist, Beijing, 100144, People's Republic of China
| | - Qian Wang
- Research Center of Plastic Surgery Hospital, Chinese Academy of Medical Sciences & Peking Union of Medical College, 33 Ba-Da-Chu Rd, Shijingshan Dist, Beijing, 100144, People's Republic of China.
| | - Jie Luan
- Breast Plastic and Reconstructive Surgery Center of Plastic Surgery Hospital, Chinese Academy of Medical Science and Peking Union Medical College, 33 Ba-Da-Chu Rd, Shijingshan Dist, Beijing, 100144, People's Republic of China.
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Leucht A, Volz AC, Rogal J, Borchers K, Kluger PJ. Advanced gelatin-based vascularization bioinks for extrusion-based bioprinting of vascularized bone equivalents. Sci Rep 2020; 10:5330. [PMID: 32210309 PMCID: PMC7093518 DOI: 10.1038/s41598-020-62166-w] [Citation(s) in RCA: 71] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Accepted: 02/27/2020] [Indexed: 12/19/2022] Open
Abstract
Bone tissue is highly vascularized. The crosstalk of vascular and osteogenic cells is not only responsible for the formation of the strongly divergent tissue types but also for their physiological maintenance and repair. Extrusion-based bioprinting presents a promising fabrication method for bone replacement. It allows for the production of large-volume constructs, which can be tailored to individual tissue defect geometries. In this study, we used the all-gelatin-based toolbox of methacryl-modified gelatin (GM), non-modified gelatin (G) and acetylated GM (GMA) to tailor both the properties of the bioink towards improved printability, and the properties of the crosslinked hydrogel towards enhanced support of vascular network formation by simple blending. The vasculogenic behavior of human dermal microvascular endothelial cells (HDMECs) and human adipose-derived stem cells (ASCs) was evaluated in the different hydrogel formulations for 14 days. Co-culture constructs including a vascular component and an osteogenic component (i.e. a bone bioink based on GM, hydroxyapatite and ASCs) were fabricated via extrusion-based bioprinting. Bioprinted co-culture constructs exhibited functional tissue-specific cells whose interplay positively affected the formation and maintenance of vascular-like structures. The setup further enabled the deposition of bone matrix associated proteins like collagen type I, fibronectin and alkaline phosphatase within the 30-day culture.
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Affiliation(s)
- A Leucht
- Institute of Interfacial Process Engineering and Plasmatechnology IGVP, University of Stuttgart, Stuttgart, Germany
- Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart, Germany
| | - A-C Volz
- Reutlingen Research Institute, Reutlingen University, Reutlingen, Germany
| | - J Rogal
- Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart, Germany
- RWTH Aachen University, Aachen, Germany
| | - K Borchers
- Institute of Interfacial Process Engineering and Plasmatechnology IGVP, University of Stuttgart, Stuttgart, Germany
- Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart, Germany
| | - P J Kluger
- Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart, Germany.
- Reutlingen Research Institute, Reutlingen University, Reutlingen, Germany.
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33
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Tissue Engineering and Regenerative Medicine in Craniofacial Reconstruction and Facial Aesthetics. J Craniofac Surg 2020; 31:15-27. [PMID: 31369496 DOI: 10.1097/scs.0000000000005840] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The craniofacial region is anatomically complex and is of critical functional and cosmetic importance, making reconstruction challenging. The limitations of current surgical options highlight the importance of developing new strategies to restore the form, function, and esthetics of missing or damaged soft tissue and skeletal tissue in the face and cranium. Regenerative medicine (RM) is an expanding field which combines the principles of tissue engineering (TE) and self-healing in the regeneration of cells, tissues, and organs, to restore their impaired function. RM offers many advantages over current treatments as tissue can be engineered for specific defects, using an unlimited supply of bioengineered resources, and does not require immunosuppression. In the craniofacial region, TE and RM are being increasingly used in preclinical and clinical studies to reconstruct bone, cartilage, soft tissue, nerves, and blood vessels. This review outlines the current progress that has been made toward the engineering of these tissues for craniofacial reconstruction and facial esthetics.
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Peng Q, Alipour H, Porsborg S, Fink T, Zachar V. Evolution of ASC Immunophenotypical Subsets During Expansion In Vitro. Int J Mol Sci 2020; 21:E1408. [PMID: 32093036 PMCID: PMC7073142 DOI: 10.3390/ijms21041408] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2020] [Revised: 02/16/2020] [Accepted: 02/17/2020] [Indexed: 12/18/2022] Open
Abstract
Adipose-derived stromal/stem cells (ASCs) are currently being considered for clinical use for a number of indications. In order to develop standardized clinical protocols, it is paramount to have a full characterization of the stem cell preparations. The surface marker expression of ASCs has previously been characterized in multiple studies. However, most of these studies have provided a cross-sectional description of ASCs in either earlier or later passages. In this study, we evaluate the dynamic changes of 15 different surface molecules during culture. Using multichromatic flow cytometry, ASCs from three different donors each in passages 1, 2, 4, 6, and 8 were analyzed for their co-expression of markers associated with mesenchymal stem cells, wound healing, immune regulation, ASC markers, and differentiation capacity, respectively. We confirmed that at an early stage, ASC displayed a high heterogeneity with a plethora of subpopulations, which by culturing became more homogeneous. After a few passages, virtually all ASCs expressed CD29, CD166 and CD201, in addition to canonical markers CD73, CD90, and CD105. However, even at passage 8, there were several predominant lineages that differed with respect to the expression of CD34, CD200 and CD271. Although the significance of remaining subpopulations still needs to be elucidated, our results underscore the necessity to fully characterize ASCs prior to clinical use.
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Affiliation(s)
| | | | | | | | - Vladimir Zachar
- Department of Health Science and Technology, Regenerative Medicine Group, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg, Denmark; (Q.P.); (H.A.); (S.P.); (T.F.)
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35
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Picke AK, Campbell GM, Blüher M, Krügel U, Schmidt FN, Tsourdi E, Winzer M, Rauner M, Vukicevic V, Busse B, Salbach-Hirsch J, Tuckermann JP, Simon JC, Anderegg U, Hofbauer LC, Saalbach A. Thy-1 (CD90) promotes bone formation and protects against obesity. Sci Transl Med 2019; 10:10/453/eaao6806. [PMID: 30089635 DOI: 10.1126/scitranslmed.aao6806] [Citation(s) in RCA: 75] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2017] [Revised: 03/02/2018] [Accepted: 06/15/2018] [Indexed: 12/19/2022]
Abstract
Osteoporosis and obesity result from disturbed osteogenic and adipogenic differentiation and present emerging challenges for our aging society. Because of the regulatory role of Thy-1 in mesenchyme-derived fibroblasts, we investigated the impact of Thy-1 expression on mesenchymal stem cell (MSC) fate between osteogenic and adipogenic differentiation and consequences for bone formation and adipose tissue development in vivo. MSCs from Thy-1-deficient mice have decreased osteoblast differentiation and increased adipogenic differentiation compared to MSCs from wild-type mice. Consistently, Thy-1-deficient mice exhibited decreased bone volume and bone formation rate with elevated cortical porosity, resulting in lower bone strength. In parallel, body weight, subcutaneous/epigonadal fat mass, and bone fat volume were increased. Thy-1 deficiency was accompanied by reduced expression of specific Wnt ligands with simultaneous increase of the Wnt inhibitors sclerostin and dickkopf-1 and an altered responsiveness to Wnt. We demonstrated that disturbed bone remodeling in osteoporosis and dysregulated adipose tissue accumulation in patients with obesity were mirrored by reduced serum Thy-1 concentrations. Our findings provide new insights into the mutual regulation of bone formation and obesity and open new perspectives to monitor and to interfere with the dysregulated balance of adipogenesis and osteogenesis in obesity and osteoporosis.
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Affiliation(s)
- Ann-Kristin Picke
- Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine III and Center for Healthy Aging, Technische Universität Dresden, Dresden 01307, Germany
| | - Graeme M Campbell
- Institute of Biomechanics, Hamburg University of Technology, 21073 Hamburg, Germany
| | | | - Ute Krügel
- Rudolf Boehm Institute of Pharmacology and Toxicology, Medical Faculty, UL, 04103 Leipzig, Germany
| | - Felix N Schmidt
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 22529 Hamburg, Germany
| | - Elena Tsourdi
- Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine III and Center for Healthy Aging, Technische Universität Dresden, Dresden 01307, Germany
| | - Maria Winzer
- Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine III and Center for Healthy Aging, Technische Universität Dresden, Dresden 01307, Germany
| | - Martina Rauner
- Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine III and Center for Healthy Aging, Technische Universität Dresden, Dresden 01307, Germany
| | - Vladimir Vukicevic
- Rudolf Boehm Institute of Pharmacology and Toxicology, Medical Faculty, UL, 04103 Leipzig, Germany
| | - Björn Busse
- Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 22529 Hamburg, Germany
| | - Juliane Salbach-Hirsch
- Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine III and Center for Healthy Aging, Technische Universität Dresden, Dresden 01307, Germany
| | - Jan P Tuckermann
- Institute of Comparative Molecular Endocrinology, Ulm University, 89081 Ulm, Germany
| | - Jan C Simon
- Department of Dermatology, Venereology and Allergology of Medical Faculty of Leipzig University, 04103 Leipzig, Germany
| | - Ulf Anderegg
- Department of Dermatology, Venereology and Allergology of Medical Faculty of Leipzig University, 04103 Leipzig, Germany
| | - Lorenz C Hofbauer
- Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine III and Center for Healthy Aging, Technische Universität Dresden, Dresden 01307, Germany
| | - Anja Saalbach
- Department of Dermatology, Venereology and Allergology of Medical Faculty of Leipzig University, 04103 Leipzig, Germany.
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36
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Kunisch E, Gunnella F, Wagner S, Dees F, Maenz S, Bossert J, Jandt KD, Kinne RW. The poly (l-lactid-co-glycolide; PLGA) fiber component of brushite-forming calcium phosphate cement induces the osteogenic differentiation of human adipose tissue-derived stem cells. ACTA ACUST UNITED AC 2019; 14:055012. [PMID: 31465298 DOI: 10.1088/1748-605x/ab3544] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
A brushite-forming calcium phosphate cement (CPC) was mechanically stabilized by addition of poly (l-lactid-co-glycolide; PLGA) fibers (≤10% w/w). It proved highly biocompatible and its fiber component enhanced bone formation in a sheep lumbar vertebroplasty model. However, possible effects on the osteogenic differentiation of resident mesenchymal stem cells (MSCs) remained unexplored. The present study used a novel approach, simultaneously analyzing the influence of a solid CPC scaffold and its relatively low PLGA proportion (a mimicry of natural bone) on osteogenic, chondrogenic, and adipogenic differentiation, as well as the pluripotency of human adipose tissue-derived mesenchymal stem cells (hASCs). hASCs were cultured on CPC discs with/without PLGA fibers (5% and 10%) in the absence of osteogenic medium for 3, 7, and 14 d. Gene expression of osteogenic markers (Runx2, osterix, alkaline phosphatase, collagen I, osteonectin, osteopontin, osteocalcin), chondrogenic markers (collagen II, Sox9, aggrecan), adipogenic markers (PPARG, Leptin, and FABP4), and pluripotency markers (Nanog, Tert, Rex) was analyzed by RT-PCR. The ability of hASCs to synthesize alkaline phosphatase was also evaluated. Cell number and viability were determined by fluorescein diacetate/propidium iodide staining. Compared to pure CPC, cultivation of hASCs on fiber-reinforced CPC transiently induced the gene expression of Runx2 and osterix (day 3), and long-lastingly augmented the expression of alkaline phosphatase (and its enzyme activity), collagen I, and osteonectin (until day 14). In contrast, augmented expression of all chondrogenic, adipogenic, and pluripotency markers was limited to day 3, followed by significant downregulation. Cultivation of hASCs on fiber-reinforced CPC reduced the cell number, but not the proportion of viable cells (viability > 95%). The PLGA component of fiber-reinforced, brushite-forming CPC supports long-lasting osteogenic differentiation of hASCs, whereas chondrogenesis, adipogenesis, and pluripotency are initially augmented, but subsequently suppressed. In view of parallel animal results, PLGA fibers may represent an interesting clinical target for future improvement of CPC- based bone regeneration.
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Affiliation(s)
- Elke Kunisch
- Experimental Rheumatology Unit, Department of Orthopedics, Jena University Hospital, Waldkliniken Eisenberg GmbH, Eisenberg, Germany
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Discussion: Adipose-Derived Stem Cells and Ceiling Culture-Derived Preadipocytes Cultured from Subcutaneous Fat Tissue Differ in Their Epigenetic Characteristics and Osteogenic Potential. Plast Reconstr Surg 2019; 144:656-657. [PMID: 31461021 DOI: 10.1097/prs.0000000000005914] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
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38
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Park KR, Yun HM, Yeo IJ, Cho S, Hong JT, Jeong YS. Peroxiredoxin 6 Inhibits Osteogenic Differentiation and Bone Formation Through Human Dental Pulp Stem Cells and Induces Delayed Bone Development. Antioxid Redox Signal 2019; 30:1969-1982. [PMID: 29792351 DOI: 10.1089/ars.2018.7530] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Aims: Peroxiredoxins (PRDXs) are thiol-specific antioxidant enzymes that regulate redox balance that are critical for maintaining the cellular potential for self-renewal and stemness. Stem cell-based regenerative medicine is a promising approach in tissue reconstruction. However, to obtain functional cells for use in clinical applications, stem cell technology still requires improvements. Results: In this study, we found that PRDX6 levels were decreased during osteogenic differentiation in human dental pulp stem cells (hDPSCs). hDPSCs stably expressing Myc-PRDX6 (hDPSC/myc-PRDX6) inhibited cell growth in hDPSCs during osteogenic differentiation and impaired osteogenic phenotypes such as alkaline phosphatase (ALP) activity, mineralized nodule formation, and osteogenic marker genes [ALP and osteocalcin (OCN)]. hDPSC cell lines stably expressing mutant glutathione peroxidase (PRDX6(C47S)) and independent phospholipase A2 (PRDX6(S32A)) were also generated. Each mutant form of PRDX6 abolished the impaired osteogenic phenotypes, the transforming growth factor-β-mediated Smad2 and p38 pathways, and RUNX2 expression. Furthermore, in vivo experiments revealed that hDPSC/myc-PRDX6 suppressed hDPSC-based bone regeneration in calvarial defect mice, and newborn PRDX6 transgenic mice exhibited delayed bone development and reduced RUNX2 expression. Innovation and Conclusion: These findings illuminate the effects of PRDX6 during osteogenic differentiation of hDPSCs, and also suggest that regulating PRDX6 may improve the clinical utility of stem cell-based regenerative medicine for the treatment of bone diseases. Antioxid. Redox Signal. 30, 1969-1982.
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Affiliation(s)
- Kyung-Ran Park
- 1 Department of Oral and Maxillofacial Regeneration, Kyung Hee University, Seoul, Republic of Korea.,2 College of Pharmacy and Medical Research Center, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
| | - Hyung-Mun Yun
- 3 Department of Oral and Maxillofacial Pathology, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea
| | - In Jun Yeo
- 2 College of Pharmacy and Medical Research Center, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
| | - Sehyung Cho
- 4 Department of Physiology, School of Medicine, Kyung Hee University, Seoul, Korea
| | - Jin Tae Hong
- 2 College of Pharmacy and Medical Research Center, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
| | - Yong Seok Jeong
- 5 Department of Biology and Research Institute of Basic Sciences, College of Sciences, Kyung Hee University, Seoul, Republic of Korea
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39
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Chen L, Tang RZ, Ruan J, Zhu XB, Yang Y. Up-regulation of THY1 attenuates interstitial pulmonary fibrosis and promotes lung fibroblast apoptosis during acute interstitial pneumonia by blockade of the WNT signaling pathway. Cell Cycle 2019; 18:670-681. [PMID: 30829553 DOI: 10.1080/15384101.2019.1578144] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Acute interstitial pneumonia (AIP) is an idiopathic pulmonary disease featuring rapid progressive dyspnea and respiratory failure. These symptoms typically develop within several days or weeks in patients without any pre-existing lung disease or external chest disease. Thymocyte differentiation antigen-1 (THY1) has been reported to have an effect on lung fibroblast proliferation and fibrogenic signaling. In this study, the mechanism of THY1 in AIP in influencing pulmonary fibrosis in terms of lung fibroblast proliferation and apoptosis was examined. An AIP mouse model with the pathological changes of lung tissues observed was established to identify the role of THY1 in the pathogenesis of AIP. The expression of THY1, a key regulator of the WNT pathway β-catenin and fibroblasts markers MMP-2, Occludin, α-SMA and Vimentin were determined. Lung fibroblasts of mice were isolated, in which THY1 expression was altered to identify roles THY1 plays in cell viability and apoptosis. A TOP/TOPflash assay was utilized to determine the activation of WNT pathway. Decrement of pulmonary fibrosis was achieved through THY1 up-regulation. The expression of MMP-2, Occludin, α-SMA, Vimentin and β-catenin, and the extent of β-catenin phosphorylation, significantly decreased, thereby indicating that THY1 overexpression inactivated WNT. Cell proliferation was inhibited and apoptosis was accelerated in lung fibroblasts transfected with vector carrying overexpressed THY1. Altogether, this study defines the potential role of THY1 in remission of AIP, via the upregulation of THY1, which renders the WNT pathway inactive. This inactivation of the WNT signaling pathway could alleviate pulmonary fibrosis by reducing lung fibroblast proliferation in AIP. Abbreviations: AIP: Acute interstitial pneumonia; ILDs: interstitial lung diseases; DAD: diffuse alveolar damage; SPF: specific-pathogen-free; NC: negative control; HCMV: human cytomegalovirus; HE: Hematoxylin-eosin; RIPA: radio-immunoprecipitation assay; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA: bovine serum albumin; HRP: horseradish peroxidase; ECL: electrochemiluminescence; FBS: fetal bovine serum; DMSO: dimethyl sulfoxide; OD: optical density.
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Affiliation(s)
- Lin Chen
- a Department of Respiratory and Critical Care Medicine , Sichuan Academy of Medical Sciences & Sichuan Province People's Hospital , Chengdu , P.R. China
| | - Rong-Zhen Tang
- b Department of Aged Infectious Diseases , Sichuan Academy of Medical Sciences & Sichuan Province People's Hospital , Chengdu , P.R. China
| | - Jia Ruan
- c Department of Respiratory Diseases , Sichuan West China Hospital Geriatric Center-Fifth People's Hospital of Sichuan Province , Chengdu , P.R. China
| | - Xiao-Bo Zhu
- d Department of Respiratory Diseases , Ziyang City People's Hospital , Ziyang , P.R. China
| | - Yang Yang
- a Department of Respiratory and Critical Care Medicine , Sichuan Academy of Medical Sciences & Sichuan Province People's Hospital , Chengdu , P.R. China
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40
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Liu S, Yang R, Yin N, Wang YL, Faiola F. Environmental and human relevant PFOS and PFOA doses alter human mesenchymal stem cell self-renewal, adipogenesis and osteogenesis. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 2019; 169:564-572. [PMID: 30476818 DOI: 10.1016/j.ecoenv.2018.11.064] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/28/2018] [Revised: 11/14/2018] [Accepted: 11/16/2018] [Indexed: 05/21/2023]
Abstract
PFOS and PFOA are two of the most abundant perfluorinated compounds (PFCs) in the environment. Previous studies have reported they have a long half-life (up to five years) once they enter into the human body. Moreover, they can potentially promote the adipogenic process by activating PPARγ. However, little is known about PFOS and PFOA chronic health impacts on humans. In this study, we employed primary human mesenchymal stem cells (hMSCs) and demonstrated that PFOS and PFOA exerted acute cytotoxicity and affected adipogenesis and osteogenesis at environmental and human relevant doses. In fact, PFOS and PFOA impaired the proper expression of CD90 (a surface antigen highly enriched in undifferentiated hMSCs) and promoted adipogenesis, presumably via their interaction with PPARγ. Moreover, PFOA partly disturbed osteogenesis. Thus, our findings not only validated the health risks of PFOS and PFOA, but also revealed new potential long-term PFOS/PFOA impacts on humans.
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Affiliation(s)
- Shuyu Liu
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China; College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Renjun Yang
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China; College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Nuoya Yin
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China; College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yuan-Liang Wang
- Department of Preventive Medicine, School of Public Health, Fujian Medical University, Fuzhou 350108, China; Section of Molecular Biology, University of California at San Diego, La Jolla, CA 92093, USA
| | - Francesco Faiola
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China; College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China.
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41
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Flores EM, Woeller CF, Falsetta ML, Susiarjo M, Phipps RP. Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis. FASEB J 2019; 33:3353-3363. [PMID: 30376360 PMCID: PMC6404567 DOI: 10.1096/fj.201801481r] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2018] [Accepted: 10/09/2018] [Indexed: 12/23/2022]
Abstract
The obesity epidemic is developing into the most costly health problem facing the world. Obesity, characterized by excessive adipogenesis and enlarged adipocytes, promotes morbidities, such as diabetes, cardiovascular disease, and cancer. Regulation of adipogenesis is critical to our understanding of how fat cell formation causes obesity and associated health problems. Thy1 (also called CD90), a widely used stem cell marker, blocks adipogenesis and reduces lipid accumulation. Thy1-knockout mice are prone to diet-induced obesity. Although the importance of Thy1 in adipogenesis and obesity is now evident, how its expression is regulated is not. We hypothesized that DNA methylation has a role in promoting adipogenesis and affects Thy1 expression. Using the methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), we investigated whether DNA methylation alters Thy1 expression during adipogenesis in both mouse 3T3-L1 preadipocytes and mouse mesenchymal stem cells. Thy1 protein and mRNA levels were decreased dramatically during adipogenesis. However, 5-aza-dC treatment prevented that phenomenon. Methylation-sensitive pyrosequencing analysis showed that CpG sites at the Thy1 locus have increased methylation during adipogenesis, as well as increased methylation in adipose tissue from diet-induced obese mice. These new findings highlight the potential role of Thy1 and DNA methylation in adipogenesis and obesity.-Flores, E. M., Woeller, C. F., Falsetta, M. L., Susiarjo, M., Phipps, R. P. Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis.
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Affiliation(s)
- E’Lissa M. Flores
- Clinical and Translational Science Institute, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA
| | - Collynn F. Woeller
- Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; and
| | - Megan L. Falsetta
- Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; and
| | - Martha Susiarjo
- Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; and
| | - Richard P. Phipps
- Clinical and Translational Science Institute, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA
- Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; and
- Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA
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42
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Saalbach A, Anderegg U. Thy‐1: more than a marker for mesenchymal stromal cells. FASEB J 2019; 33:6689-6696. [DOI: 10.1096/fj.201802224r] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Affiliation(s)
- Anja Saalbach
- Department of Dermatology, Venerology, and AllergologyFaculty of MedicineLeipzig UniversityLeipzigGermany
| | - Ulf Anderegg
- Department of Dermatology, Venerology, and AllergologyFaculty of MedicineLeipzig UniversityLeipzigGermany
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43
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Considerations for high-yield, high-throughput cell enrichment: fluorescence versus magnetic sorting. Sci Rep 2019; 9:227. [PMID: 30659223 PMCID: PMC6338736 DOI: 10.1038/s41598-018-36698-1] [Citation(s) in RCA: 107] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2018] [Accepted: 11/22/2018] [Indexed: 12/28/2022] Open
Abstract
Efficient sorting methods are required for the isolation of cellular subpopulations in basic science and translational applications. Despite this, throughputs, yields, viabilities, and processing times of common sorting methods like fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) are underreported. In the current study, we set out to quantify the ability of these sorting methods to separate defined mixtures of alkaline phosphatase liver/bone/kidney (ALPL)-expressing and non-expressing cell types. Results showed that initial MACS runs performed using manufacturer’s recommended antibody and microbead concentrations produced inaccurate ALPL+ vs. ALPL− cell splits compared to FACS when ALPL+ cells were present in larger proportions (>~25%). Accuracy at all proportions could be achieved by using substantially higher concentrations of labeling reagents. Importantly, MACS sorts resulted in only 7–9% cell loss compared to ~70% cell loss for FACS. Additionally, MACS processing was 4–6 times faster than FACS for single, low proportion samples but took similar time for single, high-proportion samples. When processing multiple samples, MACS was always faster overall due to its ability to run samples in parallel. Average cell viability for all groups remained high (>83%), regardless of sorting method. Despite requiring substantial optimization, the ability of MACS to isolate increased cell numbers in less time than FACS may prove valuable in both basic science and translational, cell-based applications.
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44
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Smith RJ, Reid AJ. The potential of adipose-derived stem cell subpopulations in regenerative medicine. Regen Med 2018; 13:357-360. [PMID: 29979099 DOI: 10.2217/rme-2018-0030] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Affiliation(s)
- Richard Jp Smith
- Blond McIndoe Laboratories, Division of Cell Matrix Biology & Regenerative Medicine, School of Biological Sciences, Faculty of Biology Medicine & Health, University of Manchester, Manchester Academic Health Science Centre, Manchester M13 9PL, UK
| | - Adam J Reid
- Blond McIndoe Laboratories, Division of Cell Matrix Biology & Regenerative Medicine, School of Biological Sciences, Faculty of Biology Medicine & Health, University of Manchester, Manchester Academic Health Science Centre, Manchester M13 9PL, UK.,Department of Plastic Surgery & Burns, Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK
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45
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Kouroupis D, Sanjurjo-Rodriguez C, Jones E, Correa D. Mesenchymal Stem Cell Functionalization for Enhanced Therapeutic Applications. TISSUE ENGINEERING PART B-REVIEWS 2018; 25:55-77. [PMID: 30165783 DOI: 10.1089/ten.teb.2018.0118] [Citation(s) in RCA: 68] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
IMPACT STATEMENT Culture expansion of MSCs has detrimental effects on various cell characteristics and attributes (e.g., phenotypic changes and senescence), which, in addition to inherent interdonor variability, negatively impact the standardization and reproducibility of their therapeutic potential. The identification of innate distinct functional MSC subpopulations, as well as the description of ex vivo protocols aimed at maintaining phenotypes and enhancing specific functions have the potential to overcome these limitations. The incorporation of those approaches into cell-based therapy would significantly impact the field, as more reproducible clinical outcomes may be achieved.
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Affiliation(s)
- Dimitrios Kouroupis
- 1 Department of Orthopedics, UHealth Sports Medicine Institute, University of Miami Miller School of Medicine, Miami, Florida.,2 Diabetes Research Institute & Cell Transplant Center, University of Miami Miller School of Medicine, Miami, Florida
| | - Clara Sanjurjo-Rodriguez
- 3 Leeds Institute of Rheumatic and Musculoskeletal Disease, Saint James University Hospital, University of Leeds, Leeds, United Kingdom.,4 Department of Biomedical Sciences, Medicine and Physiotherapy, University of A Coruña, CIBER-BBN-Institute of Biomedical Research of A Coruña (INIBIC), A Coruña, Spain
| | - Elena Jones
- 3 Leeds Institute of Rheumatic and Musculoskeletal Disease, Saint James University Hospital, University of Leeds, Leeds, United Kingdom
| | - Diego Correa
- 1 Department of Orthopedics, UHealth Sports Medicine Institute, University of Miami Miller School of Medicine, Miami, Florida.,2 Diabetes Research Institute & Cell Transplant Center, University of Miami Miller School of Medicine, Miami, Florida
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Picke AK, Campbell GM, Schmidt FN, Busse B, Rauner M, Simon JC, Anderegg U, Hofbauer LC, Saalbach A. Thy-1 Deficiency Augments Bone Loss in Obesity by Affecting Bone Formation and Resorption. Front Cell Dev Biol 2018; 6:127. [PMID: 30333974 PMCID: PMC6176687 DOI: 10.3389/fcell.2018.00127] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2018] [Accepted: 09/13/2018] [Indexed: 12/30/2022] Open
Abstract
Healthy bone remodeling results from a balanced bone formation and bone resorption realized by bone-forming osteoblasts and bone-resorbing osteoclasts, respectively. Recently, Thy-1 (CD90) was identified as positive regulator of osteoblast differentiation and activation, thus, promoting bone formation while concurrently inhibiting adipogenesis and obesity in mice. Additionally, Thy-1 did not affect bone resorption. An obesity-related co-morbidity that is increasing in prevalence is a disturbed bone formation resulting in an increased fracture risk. The underlying mechanisms of obesity-induced bone alterations are not yet fully elucidated and therefore therapy options for efficient bone-anabolic treatments are limited. Therefore, we investigated the impact of Thy-1 on bone metabolism under obese conditions. Indeed, high fat diet (HFD) induced obese mice lacking Thy-1 (Thy-1−/−) showed increased body fat mass compared to wildtype (WT) mice while bone mass (−38%) and formation (−57%) were decreased as shown by micro-computed tomography (μCT) measurement, histological analysis, and fourier-transform infrared spectroscopy (FTIR). Interestingly, under obese conditions, lack of Thy-1 affected both osteoblast and osteoclast function. Number (−30%) and activity of osteoblasts were decreased in obese Thy-1−/− mice while osteoclast number (+39%) and activity were increased. Facilitated bone marrow fat accumulation (+56%) in obese Thy-1−/− mice compared to obese WT mice was associated with upregulated tumor necrosis factor α (Tnfα, +46%) and colony stimulating factor 1 receptor (Csf1r) expression, strong promoters of osteoclast differentiation. Moreover, lack of Thy-1 was accompanied by a reduction of osteoprotegerin (Tnfrsf11b) expression (−36%), an inhibitor of osteoclast differentiation. Altered Tnfα, Csf1r, and Tnfrsf11b expression might be responsible for elevated osteoclast activity in obese Thy-1-deficient mice. In summary, our findings show that lack of Thy-1 promotes obesity under HFD conditions while concurrently decreasing bone mass and formation. Mechanistic studies revealed that under obese conditions lack of Thy-1 impairs both bone formation and bone resorption.
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Affiliation(s)
- Ann-Kristin Picke
- Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine III, Center for Healthy Aging, Technische Universität Dresden, Dresden, Germany.,Institute of Comparative Molecular Endocrinology, Ulm University, Ulm, Germany
| | - Graeme M Campbell
- Institute of Biomechanics, TUHH Hamburg University of Technology, Hamburg, Germany
| | - Felix N Schmidt
- Department of Osteology and Biomechanics, University Medical Center, Hamburg, Germany
| | - Björn Busse
- Department of Osteology and Biomechanics, University Medical Center, Hamburg, Germany
| | - Martina Rauner
- Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine III, Center for Healthy Aging, Technische Universität Dresden, Dresden, Germany
| | - Jan C Simon
- Department of Dermatology, Venerology, and Allergology, Medical Faculty, Leipzig University, Leipzig, Germany
| | - Ulf Anderegg
- Department of Dermatology, Venerology, and Allergology, Medical Faculty, Leipzig University, Leipzig, Germany
| | - Lorenz C Hofbauer
- Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine III, Center for Healthy Aging, Technische Universität Dresden, Dresden, Germany
| | - Anja Saalbach
- Department of Dermatology, Venerology, and Allergology, Medical Faculty, Leipzig University, Leipzig, Germany
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Aikawa J, Uchida K, Takano S, Inoue G, Iwase D, Miyagi M, Mukai M, Shoji S, Sekiguchi H, Takaso M. Regulation of calcitonin gene-related peptide expression through the COX-2/mPGES-1/PGE2 pathway in the infrapatellar fat pad in knee osteoarthritis. Lipids Health Dis 2018; 17:215. [PMID: 30205824 PMCID: PMC6134514 DOI: 10.1186/s12944-018-0864-8] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2018] [Accepted: 09/04/2018] [Indexed: 01/20/2023] Open
Abstract
BACKGROUND The infrapatellar fat pad (IFP) is implicated in knee osteoarthritis (KOA). Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide expressed in joint tissues and synovial tissues (ST), was recently found to be associated with KOA progression and pain. CGRP is expressed in the IFPs of human KOA patients; however, its regulation has not been elucidated. METHODS IFPs and STs were harvested from 138 KOA patients during total knee replacement (TKR) and analyzed for CGRP, cycloxygenase-2 (COX-2), and microsomal prostaglandin E synthase-1 (mPGES-1) expression using real-time polymerase chain reaction (PCR). To investigate CGRP regulation by prostaglandin E2 (PGE2), adipocytes (Ad) and the stromal vascular fraction (SVF) were harvested from IFPs using collagenase. Synovial cells (SYC) were also harvested from ST and stimulated with vehicle (serum-free culture medium), PGE2, or CGRP. RESULTS CGRP, COX-2, and mPGES-1 expression levels were significantly higher in IFPs than STs. PGE2 stimulation increased CGRP expression in Ad, the SVF, and SYC; however, CGRP expression was significantly higher in PGE2-stimulated SVF than PGE2-stimulated SYC. CGRP stimulation had no effect on COX-2 or mPGES-1 expression. CONCLUSIONS CGRP expression in the IFP of KOA patients is regulated by the COX-2/mPGES-1/PGE2 pathway.
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Affiliation(s)
- Jun Aikawa
- Department of Orthopedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-ku Kitasato, Sagamihara City, Kanagawa, 252-0374, Japan
| | - Kentaro Uchida
- Department of Orthopedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-ku Kitasato, Sagamihara City, Kanagawa, 252-0374, Japan.
| | - Shotaro Takano
- Department of Orthopedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-ku Kitasato, Sagamihara City, Kanagawa, 252-0374, Japan
| | - Gen Inoue
- Department of Orthopedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-ku Kitasato, Sagamihara City, Kanagawa, 252-0374, Japan
| | - Dai Iwase
- Department of Orthopedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-ku Kitasato, Sagamihara City, Kanagawa, 252-0374, Japan
| | - Masayuki Miyagi
- Department of Orthopedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-ku Kitasato, Sagamihara City, Kanagawa, 252-0374, Japan
| | - Manabu Mukai
- Department of Orthopedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-ku Kitasato, Sagamihara City, Kanagawa, 252-0374, Japan
| | - Shintaro Shoji
- Department of Orthopedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-ku Kitasato, Sagamihara City, Kanagawa, 252-0374, Japan
| | - Hiroyuki Sekiguchi
- Shonan University of Medical Sciences Research Institute, Nishikubo 500, Chigasaki City, Kanagawa, 253-0083, Japan
| | - Masashi Takaso
- Department of Orthopedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-ku Kitasato, Sagamihara City, Kanagawa, 252-0374, Japan
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Bothe F, Lotz B, Seebach E, Fischer J, Hesse E, Diederichs S, Richter W. Stimulation of calvarial bone healing with human bone marrow stromal cells versus inhibition with adipose-tissue stromal cells on nanostructured β-TCP-collagen. Acta Biomater 2018; 76:135-145. [PMID: 29933108 DOI: 10.1016/j.actbio.2018.06.026] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Revised: 06/11/2018] [Accepted: 06/15/2018] [Indexed: 12/28/2022]
Abstract
Bioactive functional scaffolds are essential for support of cell-based strategies to improve bone regeneration. Adipose-tissue-derived-stromal cells (ASC) are more accessible multipotent cells with faster proliferation than bone-marrow-derived-stromal-cells (BMSC) having potential to replace BMSC for therapeutic stimulation of bone-defect healing. Their osteogenic potential is, however, lower compared to BMSC, a deficit that may be overcome in growth factor-rich orthotopic bone defects with enhanced bone-conductive scaffolds. Objective of this study was to compare the therapeutic potency of human ASC and BMSC for bone regeneration on a novel nanoparticulate β-TCP/collagen-carrier (β-TNC). Cytotoxicity of β-TCP nanoparticles and multilineage differentiation of cells were characterized in vitro. Cell-seeded β-TNC versus cell-free controls were implanted into 4 mm calvarial bone-defects in immunodeficient mice and bone healing was quantified by µCT at 4 and 8 weeks. Tissue-quality and cell-origin were assessed by histology. β-TNC was non-toxic, radiolucent and biocompatible, lent excellent support for human cell persistence and allowed formation of human bone tissue by BMSC but not ASC. Opposite to BMSC, ASC-grafting significantly inhibited calvarial bone healing compared to controls. Bone formation progressed significantly from 4 to 8 weeks only in BMSC and controls yielding 5.6-fold more mineralized tissue in BMSC versus ASC-treated defects. Conclusively, β-TNC was simple to generate, biocompatible, osteoconductive, and stimulated osteogenicity of BMSC to enhance calvarial defect healing while ASC had negative effects. Thus, an orthotopic environment and β-TNC could not compensate for cell-autonomous deficits of ASC which should systematically be considered when choosing the right cell source for tissue engineering-based stimulation of bone regeneration. STATEMENT OF SIGNIFICANCE Bone-marrow-derived-stromal cells (BMSC) implanted on bone replacement materials can support bone defect healing and adipose-tissue-derived-stromal cells (ASC) being more accessible and better proliferating are considered as alternate source. This first standardized comparison of the bone regeneration potency of human ASC and BMSC was performed on a novel nanoparticular β-TCP-enriched collagen-carrier (β-TNC) designed to overcome the known inferior osteogenicity of ASC. β-TNC was non-toxic, biocompatible and osteoconductive supporting human bone formation and defect-closure by BMSC but not ASC. Long-term cell-persistence and the distinct secretome of ASC appear as main reasons why ASC inhibited bone healing opposite to BMSC. Overall, ASC-grafting is at considerable risk of producing negative effects on bone-healing while no such risks are known for BMSC.
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Fas-L promotes the stem cell potency of adipose-derived mesenchymal cells. Cell Death Dis 2018; 9:695. [PMID: 29891848 PMCID: PMC5995957 DOI: 10.1038/s41419-018-0702-y] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2018] [Revised: 04/26/2018] [Accepted: 05/14/2018] [Indexed: 12/21/2022]
Abstract
Fas-L is a TNF family member known to trigger cell death. It has recently become evident that Fas-L can transduce also non-apoptotic signals. Mesenchymal stem cells (MSCs) are multipotent cells that are derived from various adult tissues. Although MSCs from different tissues display common properties they also display tissue-specific characteristics. Previous works have demonstrated massive apoptosis following Fas-L treatment of bone marrow-derived MSCs both in vitro and following their administration in vivo. We therefore set to examine Fas-L-induced responses in adipose-derived stem cells (ASCs). Human ASCs were isolated from lipoaspirates and their reactivity to Fas-L treatment was examined. ASCs responded to Fas-L by simultaneous apoptosis and proliferation, which yielded a net doubling of cell quantities and a phenotypic shift, including reduced expression of CD105 and increased expression of CD73, in association with increased bone differentiation potential. Treatment of freshly isolated ASCs led to an increase in large colony forming unit fibroblasts, likely produced by early stem cell progenitor cells. Fas-L-induced apoptosis and proliferation signaling were found to be independent as caspase inhibition attenuated Fas-L-induced apoptosis without impacting proliferation, whereas inhibition of PI3K and MEK, but not of JNK, attenuated Fas-L-dependent proliferation, but not apoptosis. Thus, Fas-L signaling in ASCs leads to their expansion and phenotypic shift toward a more potent stem cell state. We speculate that these reactions ensure the survival of ASC progenitor cells encountering Fas-L-enriched environments during tissue damage and inflammation and may also enhance ASC survival following their administration in vivo.
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50
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Paine A, Woeller CF, Zhang H, de la Luz Garcia-Hernandez M, Huertas N, Xing L, Phipps RP, Ritchlin CT. Thy1 is a positive regulator of osteoblast differentiation and modulates bone homeostasis in obese mice. FASEB J 2018; 32:3174-3183. [PMID: 29401595 DOI: 10.1096/fj.201701379r] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Thy1 (CD90), a glycosylated, glycophosphatidylinositol-anchored membrane protein highly expressed by subsets of mesenchymal stem cells and fibroblasts, inhibits adipogenesis. The role of Thy1 on bone structure and function has been poorly studied and represents a major knowledge gap. Therefore, we analyzed the long bones of wild-type (WT) and Thy1 knockout (KO) mice with micro-computed tomography (micro-CT) and histomorphometry to compare changes in bone architecture and overall bone structure. micro-CT analysis of long bones revealed Thy1 KO and WT mice fed a high-fat diet demonstrated bone structural parameters at 4 mo that differed significantly between WT and KO mice. A significant reduction in trabecular bone volume was noted in Thy1 KO mice. The most prominent differences were observed in trabecular bone volume ratio and trabecular bone connectivity density. Consistent with micro-CT measurements, histomorphometric analysis also showed decreased bone volume in the obese Thy1 KO mice compared to obese WT mice. In vitro assays revealed that osteogenic conditions increased Thy1 expression during OB differentiation and absence of Thy1 attenuated osteoblastogenesis. Together, these findings support the concept that Thy1 serves as a major mechanistic link to regulate bone formation and negatively regulate adipogenesis.-Paine, A., Woeller, C. F., Zhang, H., Garcia-Hernandez, M. L., Huertas, N., Xing, L., Phipps, R. P., Ritchlin, C. T. Thy1 is a positive regulator of osteoblast differentiation and modulates bone homeostasis in obese mice.
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Affiliation(s)
- Ananta Paine
- Division of Allergy, Immunology, and Rheumatology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA
| | - Collynn F Woeller
- Department of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA
| | - Hengwei Zhang
- Center for Musculoskeletal Research, University of Rochester Medical Center, University of Rochester, Rochester, New York, USA; and.,Department of Pathology and Laboratory Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA
| | - Maria de la Luz Garcia-Hernandez
- Division of Allergy, Immunology, and Rheumatology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA
| | - Nelson Huertas
- Division of Allergy, Immunology, and Rheumatology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA
| | - Lianping Xing
- Center for Musculoskeletal Research, University of Rochester Medical Center, University of Rochester, Rochester, New York, USA; and.,Department of Pathology and Laboratory Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA
| | - Richard P Phipps
- Department of Environmental Medicine, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA
| | - Christopher T Ritchlin
- Division of Allergy, Immunology, and Rheumatology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA
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