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Walz S, Pollehne P, Vollmer P, Aicher WK, Stenzl A, Harland N, Amend B. Effects of Scaffolds on Urine- and Urothelial Carcinoma Tissue-Derived Organoids from Bladder Cancer Patients. Cells 2023; 12:2108. [PMID: 37626918 PMCID: PMC10453567 DOI: 10.3390/cells12162108] [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: 07/05/2023] [Revised: 08/16/2023] [Accepted: 08/17/2023] [Indexed: 08/27/2023] Open
Abstract
Organoids are three-dimensional constructs generated by placing cells in scaffolds to facilitate the growth of cultures with cell-cell and cell-matrix interactions close to the in vivo situation. Organoids may contain different types of cells, including cancer cells, progenitor cells, or differentiated cells. As distinct culture conditions have significant effects on cell metabolism, we explored the expansion of cells and expression of marker genes in bladder cancer cells expanded in two different common scaffolds. The cells were seeded in basement membrane extract (BME; s.c., Matrigel®) or in a cellulose-derived hydrogel (GrowDex®, GD) and cultured. The size of organoids and expression of marker genes were studied. We discovered that BME facilitated the growth of significantly larger organoids of cancer cell line RT112 (p < 0.05), cells from a solid tumor (p < 0.001), and a voiding urine sample (p < 0.001). Expression of proliferation marker Ki76, transcription factor TP63, cytokeratin CK20, and cell surface marker CD24 clearly differed in these different tumor cells upon expansion in BME when compared to cells in GD. We conclude that the choice of scaffold utilized for the generation of organoids has an impact not only on cell growth and organoid size but also on protein expression. The disadvantages of batch-to-batch-variations of BME must be balanced with the phenotypic bias observed with GD scaffolds when standardizing organoid cultures for clinical diagnoses.
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Affiliation(s)
- Simon Walz
- Department of Urology, University of Tuebingen Hospital, 72076 Tübingen, Germany
| | - Paul Pollehne
- Center for Medical Research, University of Tuebingen, 72074 Tübingen, Germany
| | - Philipp Vollmer
- Center for Medical Research, University of Tuebingen, 72074 Tübingen, Germany
| | - Wilhelm K. Aicher
- Center for Medical Research, University of Tuebingen, 72074 Tübingen, Germany
| | - Arnulf Stenzl
- Department of Urology, University of Tuebingen Hospital, 72076 Tübingen, Germany
| | - Niklas Harland
- Department of Urology, University of Tuebingen Hospital, 72076 Tübingen, Germany
| | - Bastian Amend
- Department of Urology, University of Tuebingen Hospital, 72076 Tübingen, Germany
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2
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Godavarthy PS, Walter CB, Lengerke C, Klein G. The Laminin Receptors Basal Cell Adhesion Molecule/Lutheran and Integrin α7β1 on Human Hematopoietic Stem Cells. Front Cell Dev Biol 2021; 9:675240. [PMID: 34746117 PMCID: PMC8570280 DOI: 10.3389/fcell.2021.675240] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2021] [Accepted: 10/01/2021] [Indexed: 11/17/2022] Open
Abstract
In the adult organism, hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow (BM) in specialized hematopoietic stem cell niches of which the extracellular matrix (ECM) is an integral component. Laminins (LM) are a family of heterotrimeric ECM molecules of which mainly family members containing an α4 or α5 chain are expressed in cells from BM niches and involved in HSPC homing and proliferation. Various integrin and non-integrin laminin receptors have been identified and characterized. Among these, the integrins α6β1 and α3β1 were reported to be strongly expressed on human and mouse HSPC. In the present study, we focus on two further specific laminin receptors, namely integrin α7β1 and basal cell adhesion molecule/Lutheran (BCAM/Lu). Using RT-PCR analyses, immunofluorescence staining, immunoblotting and flow cytometry, we show that both are strongly expressed by human lineage-negative CD34 + HSPC. Treatment with function-blocking antibodies against BCAM/Lu neither inhibits the strong adhesive interaction of CD34 + HSPC with LM-511/LM-521 nor the LM-511/LM-521 mediated changes in CD34 + HSPC proliferation, but however, influences the cytokine-induced differentiation of HSPC in colony formation assays. In addition, stromal-derived factor (SDF) 1α-mediated transmigration of CD34 + HSPC through an endothelial cell layer was effectively diminished by BCAM/Lu antibodies, suggesting a direct involvement of BCAM/Lu in the migration process. This study indicates that both laminin receptors newly identified on human CD34 + HSPC should be taken into consideration in future studies.
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Affiliation(s)
- Parimala Sonika Godavarthy
- Department of Internal Medicine II, Hematology, Oncology, Clinical Immunology and Rheumatology, University Hospital Tübingen, Tübingen, Germany
| | - Christina B Walter
- Department of Gynecology and Obstetrics, University Hospital Tübingen, Tübingen, Germany
| | - Claudia Lengerke
- Department of Internal Medicine II, Hematology, Oncology, Clinical Immunology and Rheumatology, University Hospital Tübingen, Tübingen, Germany
| | - Gerd Klein
- Department of Internal Medicine II, Hematology, Oncology, Clinical Immunology and Rheumatology, University Hospital Tübingen, Tübingen, Germany
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3
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Kumar A, Ghosh Kadamb A, Ghosh Kadamb K. Mesenchymal or Maintenance Stem Cell & Understanding Their Role in Osteoarthritis of the Knee Joint: A Review Article. THE ARCHIVES OF BONE AND JOINT SURGERY 2020; 8:560-569. [PMID: 33088856 DOI: 10.22038/abjs.2020.42536.2155] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Mesenchymal Stem Cell (MSC) therapy in osteoarthritis has been hailed as a promising treatment for osteoarthritis due to their unlimited potential of healing and regeneration. Existing literature regarding their proper name, optimal sources, mechanisms of action, dosage, and route of administration, efficacy, and safety is debatable. This index review article has tried to connect these puzzling pieces of available information and brought clarity on some of these crucial issues. The author believes that Maintenance Stem Cells (MSC) may be a more suitable term than mesenchymal stem cell or medicinal signaling cells as their origin might not be limited to mesodermal tissue. Also, they have been shown capable of self-renewal, differentiation, and maintaining a cascade of healing & possibly regeneration at the implanted site. Only a small percentage of implanted MSC survive and rest undergo apoptosis after releasing growth factors, cytokines, and extracellular vesicles. These surviving MSC become active due to conformational changes induced by anti-environment stimuli and undergo limited self-renewal, proliferation, and differentiation, but only a few of them might incorporate into the host tissues. These cells generate & maintain a momentum of series of regenerative activities to improve the function of joint, stabilize or possibly enhance the cartilage quality. More randomized studies with long term follow-up are required to bring clarity on their ideal source, expansion, culture technique, optimum dosage, and route of administration and long-term safety issues.
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Affiliation(s)
- Ashok Kumar
- Department of Orthopaedics, Saudi German Hospital, Dubai, UAE
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Temporal-controlled bioactive molecules releasing core-shell nano-system for tissue engineering strategies in endodontics. NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE 2019; 18:11-20. [PMID: 30844574 DOI: 10.1016/j.nano.2019.02.013] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/11/2018] [Revised: 02/09/2019] [Accepted: 02/13/2019] [Indexed: 01/09/2023]
Abstract
Temporal-controlled release of bioactive molecules is of key importance in the clinical translation of tissue engineering techniques. We engineered a core-shell nano-system (TD-NS) that sequentially released transforming growth factor-β1 (TGF-β1), a chemotactic/proliferating growth factor and dexamethasone (Dex), an osteo/odontogenic agent in a temporal-controlled manner. In stage-1, there was a rapid release of TGF-β1, reaching a concentration of 2 ng/mL of TGF-β1 in 7 days to 14 days, which tapers subsequently. In stage-2, Dex was released linearly from 9 days to 28 days. The TD-NS group showed a significantly higher (P < 0.05) osteo/odontogenic differentiation compared to the control and free TGF-β1 group (Free-TD) that was further corroborated with animal models/histochemical examination. The findings from this study highlighted the potential of temporal-controlled delivery of TGF-β1 and Dex from a single nano-carrier to direct spatial and temporal-control for a cell-free tissue engineering strategy in the treatment of apical periodontitis.
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Macrin D, Alghadeer A, Zhao YT, Miklas JW, Hussein AM, Detraux D, Robitaille AM, Madan A, Moon RT, Wang Y, Devi A, Mathieu J, Ruohola-Baker H. Metabolism as an early predictor of DPSCs aging. Sci Rep 2019; 9:2195. [PMID: 30778087 PMCID: PMC6379364 DOI: 10.1038/s41598-018-37489-4] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2018] [Accepted: 11/30/2018] [Indexed: 02/07/2023] Open
Abstract
Tissue resident adult stem cells are known to participate in tissue regeneration and repair that follows cell turnover, or injury. It has been well established that aging impedes the regeneration capabilities at the cellular level, but it is not clear if the different onset of stem cell aging between individuals can be predicted or prevented at an earlier stage. Here we studied the dental pulp stem cells (DPSCs), a population of adult stem cells that is known to participate in the repair of an injured tooth, and its properties can be affected by aging. The dental pulp from third molars of a diverse patient group were surgically extracted, generating cells that had a high percentage of mesenchymal stem cell markers CD29, CD44, CD146 and Stro1 and had the ability to differentiate into osteo/odontogenic and adipogenic lineages. Through RNA seq and qPCR analysis we identified homeobox protein, Barx1, as a marker for DPSCs. Furthermore, using high throughput transcriptomic and proteomic analysis we identified markers for DPSC populations with accelerated replicative senescence. In particular, we show that the transforming growth factor-beta (TGF-β) pathway and the cytoskeletal proteins are upregulated in rapid aging DPSCs, indicating a loss of stem cell characteristics and spontaneous initiation of terminal differentiation. Importantly, using metabolic flux analysis, we identified a metabolic signature for the rapid aging DPSCs, prior to manifestation of senescence phenotypes. This metabolic signature therefore can be used to predict the onset of replicative senescence. Hence, the present study identifies Barx1 as a DPSCs marker and dissects the first predictive metabolic signature for DPSCs aging.
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Affiliation(s)
- Dannie Macrin
- Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA, 98195, USA.,Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA.,Department of Genetic Engineering, SRM Institute of Science and Technology, Chennai, 603203, India
| | - Ammar Alghadeer
- Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA.,Department of Oral Health Sciences, University of Washington, School of Dentistry, Seattle, WA, 98109, USA.,Department of Biomedical Dental Sciences, Imam Abdulrahman bin Faisal University, College of Dentistry, Dammam, 31441, Saudi Arabia
| | - Yan Ting Zhao
- Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA, 98195, USA.,Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA.,Department of Oral Health Sciences, University of Washington, School of Dentistry, Seattle, WA, 98109, USA
| | - Jason W Miklas
- Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA.,Department of Bioengineering, University of Washington, Seattle, WA, 98195, USA
| | - Abdiasis M Hussein
- Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA, 98195, USA.,Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA
| | - Damien Detraux
- Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA, 98195, USA.,Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA
| | - Aaron M Robitaille
- Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA.,Department of Pharmacology, University of Washington, Seattle, WA, 98109, USA
| | - Anup Madan
- Covance Genomics Laboratory, Redmond, WA, 98052, USA
| | - Randall T Moon
- Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA.,Department of Pharmacology, University of Washington, Seattle, WA, 98109, USA
| | - Yuliang Wang
- Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA.,Paul G. Allen School of Computer Science and Engineering, University of Washington, Seattle, WA, 98195, USA
| | - Arikketh Devi
- Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA.,Department of Genetic Engineering, SRM Institute of Science and Technology, Chennai, 603203, India
| | - Julie Mathieu
- Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA, 98195, USA.,Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA.,Department of Comparative Medicine, University of Washington, School of Medicine, Seattle, WA, 98195, USA
| | - Hannele Ruohola-Baker
- Department of Biochemistry, University of Washington, School of Medicine, Seattle, WA, 98195, USA. .,Institute for Stem Cell and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, 98109, USA. .,Department of Oral Health Sciences, University of Washington, School of Dentistry, Seattle, WA, 98109, USA. .,Department of Bioengineering, University of Washington, Seattle, WA, 98195, USA.
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Odontoblast-Like Cells Differentiated from Dental Pulp Stem Cells Retain Their Phenotype after Subcultivation. Int J Cell Biol 2018; 2018:6853189. [PMID: 29670655 PMCID: PMC5836425 DOI: 10.1155/2018/6853189] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2017] [Revised: 12/08/2017] [Accepted: 01/23/2018] [Indexed: 01/07/2023] Open
Abstract
Odontoblasts, the main cell type in teeth pulp tissue, are not cultivable and they are responsible for the first line of response after dental restauration. Studies on dental materials cytotoxicity and odontoblast cells physiology require large quantity of homogenous cells retaining most of the phenotype characteristics. Odontoblast-like cells (OLC) were differentiated from human dental pulp stem cells using differentiation medium (containing TGF-β1), and OLC expanded after trypsinization (EXP-21) were evaluated and compared. Despite a slower cell growth curve, EXP-21 cells express similarly the odontoblast markers dentinal sialophosphoprotein and dentin matrix protein-1 concomitantly with RUNX2 transcripts and low alkaline phosphatase activity as expected. Both OLC and EXP-21 cells showed similar mineral deposition activity evidenced by alizarin red and von Kossa staining. These results pointed out minor changes in phenotype of subcultured EXP-21 regarding the primarily differentiated OLC, making the subcultivation of these cells a useful strategy to obtain odontoblasts for biocompatibility or cell physiology studies in dentistry.
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Park JS, Pabst AM, Ackermann M, Moergel M, Jung J, Kasaj A. Biofunctionalization of porcine-derived collagen matrix using enamel matrix derivative and platelet-rich fibrin: influence on mature endothelial cell characteristics in vitro. Clin Oral Investig 2017; 22:909-917. [PMID: 28695450 DOI: 10.1007/s00784-017-2170-7] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2016] [Accepted: 06/26/2017] [Indexed: 12/15/2022]
Abstract
OBJECTIVES The present study evaluated the effect of an enamel matrix derivative (EMD) and platelet-rich fibrin (PRF)-modified porcine-derived collagen matrix (PDCM) on human umbilical vein endothelial cells (HUVEC) in vitro. MATERIALS AND METHODS PDCM (mucoderm®) was prepared to 6 mm (±0.1 mm) diameter discs. PDCM samples were incubated with either EMD, PRF, or control solutions for 100 min at 4 °C before the experiments. Cell-inducing properties of test materials on HUVEC cells were tested with cell proliferation assays (MTT, PrestoBlue®), a cytotoxicity assay (ToxiLight®), a Boyden chamber migration assay, and a cell attachment assay. Scanning electron microscopy (SEM) imaging was performed to determine the surface and the architecture of the modified matrices. RESULTS Cell proliferation was elevated in the EMD and PRF groups compared with control (p each ≤0.046). PRF modification increased HUVEC migration ability by 8-fold compared with both control and EMD groups (p each <0.001). Both treatments significantly promoted the cell attachment of HUVEC to PDCM, as assessed by direct cell counts on the matrices (p each <0.001). CONCLUSIONS HUVEC cell characteristics were overall improved by EMD- and PRF- modified PDCM. Adsorbed bioactive molecules to the PDCM surface may have contributed to a more preferable environment to surrounding cells. CLINICAL RELEVANCE The results may give evidence that PDCM modification with EMD or PRF, respectively, might be a useful approach to improve clinical outcomes, to prevent inflammatory reactions and wound-healing disturbances, and to expand the clinical application area of PDCM.
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Affiliation(s)
- Jung Soo Park
- Department of Operative Dentistry and Periodontology, University Medical Center of the Johannes Gutenberg-University Mainz, Augustusplatz 2, 55131, Mainz, Germany
| | - Andreas Max Pabst
- Department of Oral and Maxillofacial Surgery, Federal Armed Forces Hospital, Rübenacherstr. 170, 56072, Koblenz, Germany
| | - Maximilian Ackermann
- Institute of Functional and Clinical Anatomy, University Medical Center of the Johannes Gutenberg-University Mainz, Johann-Joachim-Becher-Weg 13, 55128, Mainz, Germany
| | - Maximilian Moergel
- Department of Oral and Maxillofacial Surgery, University Medical Center of the Johannes Gutenberg-University Mainz, Augustusplatz 2, 55131, Mainz, Germany
| | - Junho Jung
- Department of Oral and Maxillofacial Surgery, University Medical Center of the Johannes Gutenberg-University Mainz, Augustusplatz 2, 55131, Mainz, Germany
| | - Adrian Kasaj
- Department of Operative Dentistry and Periodontology, University Medical Center of the Johannes Gutenberg-University Mainz, Augustusplatz 2, 55131, Mainz, Germany.
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8
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Mesenchymal stem cell detachment with trace trypsin is superior to EDTA for in vitro chemotaxis and adhesion assays. Biochem Biophys Res Commun 2017; 484:656-661. [PMID: 28153723 DOI: 10.1016/j.bbrc.2017.01.171] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2017] [Accepted: 01/28/2017] [Indexed: 02/06/2023]
Abstract
Trypsin is frequently used to dissociate mesenchymal stem cells (MSCs) for in vitro adhesion and chemotaxis assays. However, its potential impact on surface receptor degradation is poorly understood. The purpose of this study was to evaluate the effect of trypsin-EDTA exposure versus PBS-EDTA on MSC surface receptor integrity and function. Primary human MSCs were detached with PBS-EDTA alone, or Cell Dissociation Buffer followed by 30 s exposure to 0.05% w/v trypsin-EDTA (trace trypsin method, TT), or 0.25% w/v trypsin exposure for 2 or 5 min. Cells were characterized for surface integrity of β1 integrin (CD29) and PDGF Receptor (PDGF-R), and assessed in vitro for adhesion to atelocollagen-coated surfaces and migration to PDGF-BB. PBS-EDTA detachment fully preserved receptor integrity but routinely detached only half of the adherent cells and led to cell aggregates that failed to adhere evenly across the Transwell migration insert. Both CD29 and PDGF-R were significantly degraded by 0.25% trypsin detachment for 2 or 5 min compared to the TT method or PBS-EDTA (p < 0.05). Cells migrated optimally to PDGF-BB when detached with the TT method (3.1-fold vs α-MEM, p = 0.01). Cells attached optimally to atelocollagen when detached using the TT method or PBS-EDTA (6- to 10-fold vs 0.25% trypsin, p < 0.01). CDB followed by trace trypsin-EDTA exposure is recommended over PBS-EDTA to produce a single-cell MSC suspension that preserves receptor integrity and more reproducible receptor-mediated responses.
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Dan P, Velot É, Francius G, Menu P, Decot V. Human-derived extracellular matrix from Wharton's jelly: An untapped substrate to build up a standardized and homogeneous coating for vascular engineering. Acta Biomater 2017; 48:227-237. [PMID: 27769940 DOI: 10.1016/j.actbio.2016.10.018] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2016] [Revised: 10/04/2016] [Accepted: 10/11/2016] [Indexed: 12/18/2022]
Abstract
One of the outstanding goals in tissue engineering is to develop a natural coating surface which is easy to manipulate, effective for cell adhesion and fully biocompatible. The ideal surface would be derived from human tissue, perfectly controllable, and pathogen-free, thereby satisfying all of the standards of the health authorities. This paper reports an innovative approach to coating surfaces using a natural extracellular matrix (ECM) extracted from the Wharton's jelly (WJ) of the umbilical cord (referred to as WJ-ECM). We have shown by atomic force microscopy (AFM), that the deposition of WJ-ECM on surfaces is homogenous with a controllable thickness, and that this easily-prepared coating is appropriate for both the adhesion and proliferation of human mesenchymal stem cells and mature endothelial cells. Furthermore, under physiological shear stress conditions, a larger number of cells remained adhered to WJ-ECM than to a conventional coating such as collagen - a result supported by the higher expression of both integrins α2 and β1 in cells cultured on WJ-ECM. Our data clearly show that Wharton's jelly is a highly promising coating for the design of human biocompatible surfaces in tissue engineering as well as in regenerative medicine. STATEMENT OF SIGNIFICANCE Discovery and design of biomaterial surface are a hot spot in the tissue engineering field. Natural matrix is preferred to mimic native cell microenvironment but its use is limited due to poor resource availability. Moreover, current studies often use single or several components of natural polymers, which is not the case in human body. This paper reports a natural extracellular matrix with full components derived from healthy human tissue: Wharton's jelly of umbilical cord. Reconstituting this matrix as a culture surface, our easily-prepared coating provides superior biocompatibility for stem and mature cells. Furthermore, we observed improved cell performance on this coating under both static and dynamic condition. This novel human derived ECM would be a promising choice for regenerative medicine.
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Affiliation(s)
- Pan Dan
- UMR 7365 CNRS, Ingénierie Moléculaire et Physiopathologie Articulaire Université de Lorraine, Vandœuvre-lès-Nancy Cedex 54505, France
| | - Émilie Velot
- UMR 7365 CNRS, Ingénierie Moléculaire et Physiopathologie Articulaire Université de Lorraine, Vandœuvre-lès-Nancy Cedex 54505, France
| | - Grégory Francius
- UMR 7564, Université de Lorraine, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, Villers-lès-Nancy 54600, France
| | - Patrick Menu
- UMR 7365 CNRS, Ingénierie Moléculaire et Physiopathologie Articulaire Université de Lorraine, Vandœuvre-lès-Nancy Cedex 54505, France.
| | - Véronique Decot
- UMR 7365 CNRS, Ingénierie Moléculaire et Physiopathologie Articulaire Université de Lorraine, Vandœuvre-lès-Nancy Cedex 54505, France; Unité de Thérapie Cellulaire et Tissulaire, CHRU de Nancy, Vandœuvre-lès-Nancy 54511, France
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Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium. Stem Cells Int 2016; 2016:5246584. [PMID: 27006663 PMCID: PMC4781990 DOI: 10.1155/2016/5246584] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2015] [Revised: 01/18/2016] [Accepted: 01/26/2016] [Indexed: 11/17/2022] Open
Abstract
The manufacture of human mesenchymal stem cells (hMSCs) for clinical applications requires an appropriate growth surface and an optimized, preferably chemically defined medium (CDM) for expansion. We investigated a new protein/peptide-free CDM that supports the adhesion, growth, and detachment of an immortalized hMSC line (hMSC-TERT) as well as primary cells derived from bone marrow (bm-hMSCs) and adipose tissue (ad-hMSCs). We observed the rapid attachment and spreading of hMSC-TERT cells and ad-hMSCs in CDM concomitant with the expression of integrin and actin fibers. Cell spreading was promoted by coating the growth surface with collagen type IV and fibronectin. The growth of hMSC-TERT cells was similar in CDM and serum-containing medium whereas the lag phase of bm-hMSCs was prolonged in CDM. FGF-2 or surface coating with collagen type IV promoted the growth of bm-hMSCs, but laminin had no effect. All three cell types retained their trilineage differentiation capability in CDM and were detached by several enzymes (but not collagenase in the case of hMSC-TERT cells). The medium and coating did not affect detachment efficiency but influenced cell survival after detachment. CDM combined with cell-specific surface coatings and/or FGF-2 supplements is therefore as effective as serum-containing medium for the manufacture of different hMSC types.
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11
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Maerz JK, Roncoroni LP, Goldeck D, Abruzzese T, Kalbacher H, Rolauffs B, DeZwart P, Nieselt K, Hart ML, Klein G, Aicher WK. Bone marrow-derived mesenchymal stromal cells differ in their attachment to fibronectin-derived peptides from term placenta-derived mesenchymal stromal cells. Stem Cell Res Ther 2016; 7:29. [PMID: 26869043 PMCID: PMC4751672 DOI: 10.1186/s13287-015-0243-6] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2015] [Revised: 11/03/2015] [Accepted: 11/18/2015] [Indexed: 12/17/2022] Open
Abstract
INTRODUCTION Human mesenchymal stromal cells (MSCs) can be isolated from different sources including bone marrow and term placenta. These two populations display distinct patterns of proliferation and differentiation in vitro. Since proliferation and differentiation of cells are modulated by cell-matrix interactions, we investigated the attachment of MSCs to a set of peptide-coated surfaces and explored their interactions with peptides in suspension. METHODS Human MSCs were isolated from bone marrow and term placenta and expanded. Binding of MSCs to peptides was investigated by a cell-attachment spot assay, by blocking experiments and flow cytometry. The integrin expression pattern was explored by a transcript array and corroborated by quantitative reverse transcription polymerase chain reaction and flow cytometry. RESULTS Expanded placenta-derived MSCs (pMSCs) attached well to surfaces coated with fibronectin-derived peptides P7, P15, and P17, whereas bone marrow-derived MSCs (bmMSCs) attached to P7, but barely to P15 and P17. The binding of bmMSCs and pMSCs to the peptides was mediated by β1 integrins. In suspension, expanded bmMSCs barely bind to P7, P13, P15, and less to P14 and P17. Ex vivo, bmMSCs failed to bind P7, but displayed a weak interaction with P13, P14, and P15. In suspension, expanded pMSCs displayed binding to many peptides, including P4, P7, P13, P14, P15, and P17. The differences observed in binding of bmMSCs and pMSCs to the peptides were associated with significant differences in expression of integrin α2-, α4-, and α6-chains. CONCLUSIONS Human bmMSCs and pMSCs show distinct patterns of attachment to defined peptides and maintain differences in expression of integrins in vitro. Interactions of ex vivo bmMSCs with a given peptide yield different staining patterns compared to expanded bmMSCs in suspension. Attachment of expanded MSCs to peptides on surfaces is different from interactions of expanded MSCs with peptides in suspension. Studies designed to investigate the interactions of human MSCs with peptide-augmented scaffolds or peptides in suspension must therefore regard these differences in cell-peptide interactions.
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Affiliation(s)
- Jan K Maerz
- KFO273, Department of Urology, University of Tübingen Hospital, Paul Ehrlich Str. 15, 72076, Tübingen, Germany.
| | - Lorenzo P Roncoroni
- KFO273, Department of Urology, University of Tübingen Hospital, Paul Ehrlich Str. 15, 72076, Tübingen, Germany.
| | - David Goldeck
- Center for Medical Research, Department of Medicine II, University of Tübingen, Tübingen, Germany.
| | - Tanja Abruzzese
- KFO273, Department of Urology, University of Tübingen Hospital, Paul Ehrlich Str. 15, 72076, Tübingen, Germany.
| | - Hubert Kalbacher
- Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.
| | - Bernd Rolauffs
- BG Trauma Center Tübingen, University of Tübingen, Tübingen, Germany.
| | - Peter DeZwart
- BG Trauma Center Tübingen, University of Tübingen, Tübingen, Germany.
| | - Kay Nieselt
- Integrative Transcriptomics, Center for Bioinformatics, University of Tübingen, Tübingen, Germany.
| | - Melanie L Hart
- KFO273, Department of Urology, University of Tübingen Hospital, Paul Ehrlich Str. 15, 72076, Tübingen, Germany.
| | - Gerd Klein
- Center for Medical Research, Department of Medicine II, University of Tübingen, Tübingen, Germany.
| | - Wilhelm K Aicher
- KFO273, Department of Urology, University of Tübingen Hospital, Paul Ehrlich Str. 15, 72076, Tübingen, Germany.
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12
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McKayed K, Prendergast PJ, Campbell VA. Aging enhances the vulnerability of mesenchymal stromal cells to uniaxial tensile strain-induced apoptosis. J Biomech 2016; 49:458-62. [DOI: 10.1016/j.jbiomech.2015.11.053] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2015] [Revised: 11/18/2015] [Accepted: 11/21/2015] [Indexed: 10/22/2022]
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13
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Human Mesenchymal Stromal Cells from Different Sources Diverge in Their Expression of Cell Surface Proteins and Display Distinct Differentiation Patterns. Stem Cells Int 2015; 2016:5646384. [PMID: 26770208 PMCID: PMC4684891 DOI: 10.1155/2016/5646384] [Citation(s) in RCA: 129] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2015] [Revised: 08/03/2015] [Accepted: 08/03/2015] [Indexed: 12/13/2022] Open
Abstract
When germ-free cell cultures became a laboratory routine, hopes were high for using this novel technology for treatment of diseases or replacement of cells in patients suffering from injury, inflammation, or cancer or even refreshing cells in the elderly. Today, more than 50 years after the first successful bone marrow transplantation, clinical application of hematopoietic stem cells is a routine procedure, saving the lives of many every day. However, transplanting other than hematopoietic stem and progenitor cells is still limited to a few applications, and it mainly applies to mesenchymal stromal cells (MSCs) isolated from bone marrow. But research progressed and different trials explore the clinical potential of human MSCs isolated from bone marrow but also from other tissues including adipose tissue. Recently, MSCs isolated from bone marrow (bmMSCs) were shown to be a blend of distinct cells and MSCs isolated from different tissues show besides some common features also some significant differences. This includes the expression of distinct antigens on subsets of MSCs, which was utilized recently to define and separate functionally different subsets from bulk MSCs. We therefore briefly discuss differences found in subsets of human bmMSCs and in MSCs isolated from some other sources and touch upon how this could be utilized for cell-based therapies.
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Noh MJ, Lee KH. Orthopedic cellular therapy: An overview with focus on clinical trials. World J Orthop 2015; 6:754-61. [PMID: 26601056 PMCID: PMC4644862 DOI: 10.5312/wjo.v6.i10.754] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/22/2015] [Revised: 08/22/2015] [Accepted: 09/25/2015] [Indexed: 02/06/2023] Open
Abstract
In this editorial, the authors tried to evaluate the present state of cellular therapy in orthopedic field. The topics the authors try to cover include not only the clinical trials but the various research areas as well. Both the target diseases for cellular therapy and the target cells were reviewed. New methods to activate the cells were interesting to review. Most advanced clinical trials were also included because several of them have advanced to phase III clinical trials. In the orthopedic field, there are many diseases with a definite treatment gap at this time. Because cellular therapies can regenerate damaged tissues, there is a possibility for cellular therapies to become disease modifying drugs. It is not clear whether cellular therapies will become the standard of care in any of the orthopedic disorders, however the amount of research being performed and the number of clinical trials that are on-going make the authors believe that cellular therapies will become important treatment modalities within several years.
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Ulrich C, Abruzzese T, Maerz JK, Ruh M, Amend B, Benz K, Rolauffs B, Abele H, Hart ML, Aicher WK. Human Placenta-Derived CD146-Positive Mesenchymal Stromal Cells Display a Distinct Osteogenic Differentiation Potential. Stem Cells Dev 2015; 24:1558-69. [DOI: 10.1089/scd.2014.0465] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Affiliation(s)
- Christine Ulrich
- Center for Regenerative Medicine, University of Tübingen Hospital, Tübingen, Germany
| | - Tanja Abruzzese
- Department of Urology, University of Tübingen Hospital, Tübingen, Germany
| | - Jan K. Maerz
- Center for Regenerative Medicine, University of Tübingen Hospital, Tübingen, Germany
| | - Manuel Ruh
- Center for Regenerative Medicine, University of Tübingen Hospital, Tübingen, Germany
| | - Bastian Amend
- Department of Urology, University of Tübingen Hospital, Tübingen, Germany
| | - Karin Benz
- NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany
| | - Bernd Rolauffs
- Department of Traumatology, BGU Hospital, University of Tübingen, Tübingen, Germany
| | - Harald Abele
- Department of Gynecology and Obstetrics, University of Tübingen Hospital, Tübingen, Germany
| | - Melanie L. Hart
- Department of Urology, University of Tübingen Hospital, Tübingen, Germany
| | - Wilhelm K. Aicher
- Center for Regenerative Medicine, University of Tübingen Hospital, Tübingen, Germany
- Department of Urology, University of Tübingen Hospital, Tübingen, Germany
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16
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Crabbé A, Liu Y, Sarker SF, Bonenfant NR, Barrila J, Borg ZD, Lee JJ, Weiss DJ, Nickerson CA. Recellularization of decellularized lung scaffolds is enhanced by dynamic suspension culture. PLoS One 2015; 10:e0126846. [PMID: 25962111 PMCID: PMC4427280 DOI: 10.1371/journal.pone.0126846] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2014] [Accepted: 04/08/2015] [Indexed: 12/20/2022] Open
Abstract
Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.
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Affiliation(s)
- Aurélie Crabbé
- The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, Arizona, United States of America
| | - Yulong Liu
- The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, Arizona, United States of America
| | - Shameema F. Sarker
- The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, Arizona, United States of America
| | - Nicholas R. Bonenfant
- Department of Medicine, University of Vermont College of Medicine, Burlington, Vermont, United States of America
| | - Jennifer Barrila
- The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, Arizona, United States of America
| | - Zachary D. Borg
- Department of Medicine, University of Vermont College of Medicine, Burlington, Vermont, United States of America
| | - James J. Lee
- Division of Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Mayo Clinic Arizona, Scottsdale, Arizona, United States of America
| | - Daniel J. Weiss
- Department of Medicine, University of Vermont College of Medicine, Burlington, Vermont, United States of America
| | - Cheryl A. Nickerson
- The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, Arizona, United States of America
- School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America
- * E-mail:
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17
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von Erlach TC, Hedegaard MAB, Stevens MM. High resolution Raman spectroscopy mapping of stem cell micropatterns. Analyst 2015; 140:1798-803. [PMID: 25671676 PMCID: PMC5407440 DOI: 10.1039/c4an02346c] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
We report on the use of high resolution Raman spectroscopy mapping combined with a micro-engineered stem cell platform. This technique obtains quantitative information about the concentration of individual intracellular molecules such as proteins, lipids, and other metabolites, while tightly controlling cell shape and adhesion. This new quantitative analysis will prove highly relevant for in vitro drug screening applications and regenerative medicine.
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Affiliation(s)
- Thomas C von Erlach
- Department of Materials, Department of Bioengineering and Institute of Biomedical Engineering, Imperial College London, London SW7 2AZ, UK.
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18
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Kim J, Lin B, Kim S, Choi B, Evseenko D, Lee M. TGF-β1 conjugated chitosan collagen hydrogels induce chondrogenic differentiation of human synovium-derived stem cells. J Biol Eng 2015; 9:1. [PMID: 25745515 PMCID: PMC4350967 DOI: 10.1186/1754-1611-9-1] [Citation(s) in RCA: 78] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2014] [Accepted: 12/24/2014] [Indexed: 12/27/2022] Open
Abstract
Background Unlike bone tissue, articular cartilage regeneration has not been very successful and has many challenges ahead. We have previously developed injectable hydrogels using photopolymerizable chitosan (MeGC) that supported growth of chondrocytes. In this study, we demonstrate a biofunctional hydrogel for specific use in cartilage regeneration by conjugating transforming growth factor-β1 (TGF-β1), a well-documented chondrogenic factor, to MeGC hydrogels impregnating type II collagen (Col II), one of the major cartilaginous extracellular matrix (ECM) components. Results TGF-β1 was delivered from MeGC hydrogels in a controlled manner with reduced burst release by chemically conjugating the protein to MeGC. The hydrogel system did not compromise viability of encapsulated human synovium-derived mesenchymal stem cells (hSMSCs). Col II impregnation and TGF-β1 delivery significantly enhanced cellular aggregation and deposition of cartilaginous ECM by the encapsulated cells, compared with pure MeGC hydrogels. Conclusions This study demonstrates successful engineering of a biofunctional hydrogel with a specific microenvironment tailored to promote chondrogenesis. This hydrogel system can provide promising efficacious therapeutics in the treatment of cartilage defects. Electronic supplementary material The online version of this article (doi:10.1186/1754-1611-9-1) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Jinku Kim
- Department of Bio and Chemical Engineering, Hongik University, Sejong, 339-701 South Korea
| | - Brian Lin
- Division of Advanced Prosthodontics, University of California, Los Angeles, CA 90095 USA
| | - Soyon Kim
- Department of Bioengineering, University of California, Los Angeles, CA 90095 USA
| | - Bogyu Choi
- Division of Advanced Prosthodontics, University of California, Los Angeles, CA 90095 USA
| | - Denis Evseenko
- Department of Orthopaedic Surgery, University of California, Los Angeles, CA 90095 USA
| | - Min Lee
- Division of Advanced Prosthodontics, University of California, Los Angeles, CA 90095 USA ; Department of Bioengineering, University of California, Los Angeles, CA 90095 USA
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19
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Ding L, Li X, Sun H, Su J, Lin N, Péault B, Song T, Yang J, Dai J, Hu Y. Transplantation of bone marrow mesenchymal stem cells on collagen scaffolds for the functional regeneration of injured rat uterus. Biomaterials 2014; 35:4888-4900. [PMID: 24680661 DOI: 10.1016/j.biomaterials.2014.02.046] [Citation(s) in RCA: 167] [Impact Index Per Article: 15.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2014] [Accepted: 02/23/2014] [Indexed: 02/06/2023]
Abstract
Serious injuries of endometrium in women of reproductive age are often followed by uterine scar formation and a lack of functional endometrium predisposing to infertility or miscarriage. Bone marrow-derived mesenchymal stem cells (BM-MSCs) have shown great promise in clinical applications. In the present study, BM-MSCs loaded onto degradable collagen membranes were constructed. Collagen membranes provided 3-dimmensional architecture for the attachment, growth and migration of rat BM-MSCs and did not impair the expression of the stemness genes. We then investigated the effect of collagen/BM-MSCs constructs in the healing of severe uterine injury in rats (partial full thickness uterine excision). At four weeks after the transplantation of collagen/BM-MSCs constructs, BM-MSCs were mainly located to the basal membrane of regenerative endometrium. The wounded tissue adjacent to collagen/BM-MSCs constructs expressed higher level of bFGF, IGF-1, TGFβ1 and VEGF than the corresponding tissue in rats receiving collagen construct alone or in spontaneous regeneration group. Moreover, the collagen/BM-MSCs system increased proliferative abilities of uterine endometrial and muscular cells, facilitated microvasculature regeneration, and restored the ability of endometrium to receive the embryo and support its development to a viable stage. Our findings indicate that BM-MSCs may support uterine tissue regeneration.
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Affiliation(s)
- Lijun Ding
- Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Rd., Nanjing 210008, China; Nanjing Center for Stem Cells and Biomaterials, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Rd., Nanjing 210008, China
| | - Xin'an Li
- Department of Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Rd., Nanjing 210008, China
| | - Haixiang Sun
- Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Rd., Nanjing 210008, China
| | - Jing Su
- Department of Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Rd., Nanjing 210008, China
| | - Nacheng Lin
- Department of Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Rd., Nanjing 210008, China
| | - Bruno Péault
- Center for Regenerative Medicine, University of Edinburgh, Edinburgh, EH16 4UU, UK
| | - Tianran Song
- Department of Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Rd., Nanjing 210008, China
| | - Jun Yang
- Department of Pathology, Nanjing Drum Tower Hospital Affiliated to Nanjing University Medical School, 321 Zhongshan Rd. Nanjing 210008, China
| | - Jianwu Dai
- Nanjing Center for Stem Cells and Biomaterials, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Rd., Nanjing 210008, China; Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 3 Nanyitiao, Zhongguancun, Beijing 100190, China.
| | - Yali Hu
- Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Rd., Nanjing 210008, China; Nanjing Center for Stem Cells and Biomaterials, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Rd., Nanjing 210008, China; Department of Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 321 Zhongshan Rd., Nanjing 210008, China.
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20
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Ghosh D, Lili L, McGrail DJ, Matyunina LV, McDonald JF, Dawson MR. Integral role of platelet-derived growth factor in mediating transforming growth factor-β1-dependent mesenchymal stem cell stiffening. Stem Cells Dev 2014; 23:245-61. [PMID: 24093435 PMCID: PMC3904528 DOI: 10.1089/scd.2013.0240] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2013] [Accepted: 10/04/2013] [Indexed: 12/20/2022] Open
Abstract
Mesenchymal stem cells (MSCs) play an important role in matrix remodeling, fibroblast activation, angiogenesis, and immunomodulation and are an integral part of fibrovascular networks that form in developing tissues and tumors. The engraftment and function of MSCs in tissue niches is regulated by a multitude of soluble proteins. Transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor-BB (PDGF) have previously been recognized for their role in MSC biology; thus, we sought to investigate their function in mediating MSC mechanics and matrix interactions. Cytoskeletal organization, characterized by cell elongation, stress fiber formation, and condensation of actin and microtubules, was dramatically affected by TGF-β1, individually and in combination with PDGF. The intracellular mechanical response to these stimuli was measured with particle tracking microrheology. MSCs stiffened in response to TGF-β1 (their elastic moduli was ninefold higher than control cells), a result that was enhanced by the addition of PDGF (100-fold change). Blocking TGF-β1 or PDGF signaling with inhibitors SB-505124 or JNJ-10198409, respectively, reversed soluble-factor-induced stiffening, indicating that crosstalk between these two pathways is essential for stiffening response. A genome-wide microarray analysis revealed TGF-β1-dependent regulation of cytoskeletal actin-binding protein genes. Actin crosslinking and bundling protein genes, which regulate cytosolic rheology through changes in semiflexible actin polymer meshwork, were upregulated with TGF-β1 treatment. TGF-β1 alone and in combination with PDGF also amplified surface integrin expression and adhesivity of MSCs with extracellular matrix proteins. These findings will provide a more mechanistic insight for modeling tissue-level rigidity in fibrotic tissues and tumors.
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Affiliation(s)
- Deepraj Ghosh
- Georgia Institute of Technology, School of Chemical and Biomolecular Engineering, Atlanta, Georgia
| | - Loukia Lili
- Georgia Institute of Technology, School of Biology, Atlanta, Georgia
- Georgia Institute of Technology, Integrated Cancer Research Center, Atlanta, Georgia
| | - Daniel J. McGrail
- Georgia Institute of Technology, School of Chemical and Biomolecular Engineering, Atlanta, Georgia
| | - Lilya V. Matyunina
- Georgia Institute of Technology, School of Biology, Atlanta, Georgia
- Georgia Institute of Technology, Integrated Cancer Research Center, Atlanta, Georgia
| | - John F. McDonald
- Georgia Institute of Technology, School of Biology, Atlanta, Georgia
- Georgia Institute of Technology, Integrated Cancer Research Center, Atlanta, Georgia
- The Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia
| | - Michelle R. Dawson
- Georgia Institute of Technology, School of Chemical and Biomolecular Engineering, Atlanta, Georgia
- The Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia
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21
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Chiu LH, Lai WFT, Chang SF, Wong CC, Fan CY, Fang CL, Tsai YH. The effect of type II collagen on MSC osteogenic differentiation and bone defect repair. Biomaterials 2014; 35:2680-91. [PMID: 24411332 DOI: 10.1016/j.biomaterials.2013.12.005] [Citation(s) in RCA: 89] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2013] [Accepted: 12/08/2013] [Indexed: 01/27/2023]
Abstract
The function of type II collagen in cartilage is well documented and its importance for long bone development has been implicated. However, the involvement of type II collagen in bone marrow derived mesenchymal stem cell (BMSC) osteogenesis has not been well investigated. This study elucidated the pivotal role of type II collagen in BMSC osteogenesis and its potential application to bone healing. Type II collagen-coated surface was found to accelerate calcium deposition, and the interaction of osteogenic medium-induced BMSCs with type II collagen-coated surface was mainly mediated through integrin α2β1. Exogenous type II collagen directly activated FAK-JNK signaling and resulted in the phosphorylation of RUNX2. In a segmental defect model in rats, type II collagen-HA/TCP-implanted rats showed significant callus formation at the reunion site, and a higher SFI (sciatic function index) scoring as comparing to other groups were also observed at 7, 14, and 21 day post-surgery. Collectively, type II collagen serves as a better modulator during early osteogenic differentiation of BMSCs by facilitating RUNX2 activation through integrin α2β1-FAK-JNK signaling axis, and enhance bone defect repair through an endochondral ossification-like process. These results advance our understanding about the cartilaginous ECM-BMSC interaction, and provide perspective for bone defect repair strategies.
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Affiliation(s)
- Li-Hsuan Chiu
- Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 11031, Taiwan
| | - Wen-Fu T Lai
- Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 11031, Taiwan; Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei 11031, Taiwan; Center for Nano Biomedicine Research, Taipei Medical University, Taipei 11031, Taiwan
| | - Shwu-Fen Chang
- Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 11031, Taiwan
| | - Chin-Chean Wong
- Department of Orthopaedic Surgery, Wanfang Hospital, Taipei Medical University, Taipei 11031, Taiwan
| | - Cheng-Yu Fan
- Department of Orthopaedic Surgery, Taipei Medical University Hospital, Taipei 11031, Taiwan
| | - Chia-Lang Fang
- Department of Pathology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan
| | - Yu-Hui Tsai
- Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 11031, Taiwan; Center for Nano Biomedicine Research, Taipei Medical University, Taipei 11031, Taiwan.
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22
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MacQueen L, Sun Y, Simmons CA. Mesenchymal stem cell mechanobiology and emerging experimental platforms. J R Soc Interface 2013; 10:20130179. [PMID: 23635493 PMCID: PMC3673151 DOI: 10.1098/rsif.2013.0179] [Citation(s) in RCA: 101] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2013] [Accepted: 04/10/2013] [Indexed: 12/18/2022] Open
Abstract
Experimental control over progenitor cell lineage specification can be achieved by modulating properties of the cell's microenvironment. These include physical properties of the cell adhesion substrate, such as rigidity, topography and deformation owing to dynamic mechanical forces. Multipotent mesenchymal stem cells (MSCs) generate contractile forces to sense and remodel their extracellular microenvironments and thereby obtain information that directs broad aspects of MSC function, including lineage specification. Various physical factors are important regulators of MSC function, but improved understanding of MSC mechanobiology requires novel experimental platforms. Engineers are bridging this gap by developing tools to control mechanical factors with improved precision and throughput, thereby enabling biological investigation of mechanics-driven MSC function. In this review, we introduce MSC mechanobiology and review emerging cell culture platforms that enable new insights into mechanobiological control of MSCs. Our main goals are to provide engineers and microtechnology developers with an up-to-date description of MSC mechanobiology that is relevant to the design of experimental platforms and to introduce biologists to these emerging platforms.
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Affiliation(s)
- Luke MacQueen
- Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario, Canada
| | - Yu Sun
- Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario, Canada
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
| | - Craig A. Simmons
- Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario, Canada
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
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23
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Yang D, Lü X, Hong Y, Xi T, Zhang D. The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials. Biomaterials 2013; 34:5747-58. [DOI: 10.1016/j.biomaterials.2013.04.028] [Citation(s) in RCA: 75] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2013] [Accepted: 04/13/2013] [Indexed: 12/17/2022]
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Human endothelial-like differentiated precursor cells maintain their endothelial characteristics when cocultured with mesenchymal stem cell and seeded onto human cancellous bone. Mediators Inflamm 2013; 2013:364591. [PMID: 23476102 PMCID: PMC3588182 DOI: 10.1155/2013/364591] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2012] [Revised: 12/20/2012] [Accepted: 01/03/2013] [Indexed: 12/15/2022] Open
Abstract
Introduction. Cancellous bone is frequently used for filling bone defects in a clinical setting. It provides favourable conditions for regenerative cells such as MSC and early EPC. The combination of MSC and EPC results in superior bone healing in experimental bone healing models. Materials and Methods. We investigated the influence of osteogenic culture conditions on the endothelial properties of early EPC and the osteogenic properties of MSC when cocultured on cancellous bone. Additionally, cell adhesion, metabolic activity, and differentiation were assessed 2, 6, and 10 days after seeding. Results. The number of adhering EPC and MSC decreased over time; however the cells remained metabolically active over the 10-day measurement period. In spite of a decline of lineage specific markers, cells maintained their differentiation to a reduced level. Osteogenic stimulation of EPC caused a decline but not abolishment of endothelial characteristics and did not induce osteogenic gene expression. Osteogenic stimulation of MSC significantly increased their metabolic activity whereas collagen-1α and alkaline phosphatase gene expressions declined. When cocultured with EPC, MSC's collagen-1α gene expression increased significantly. Conclusion. EPC and MSC can be cocultured in vitro on cancellous bone under osteogenic conditions, and coculturing EPC with MSC stabilizes the latter's collagen-1α gene expression.
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25
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Nor FM, Kurniawan D, Seo YK, Park JK, Lee HY, Lim JY. Polycaprolactone–starch blends with corn-based coupling agent: physical properties and in vitro analysis. Proc Inst Mech Eng H 2012; 226:693-8. [DOI: 10.1177/0954411912452988] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
In an attempt to improve properties of polycaprolcatone–starch blend, this study uses zein as coupling agent in preparing the blend through a single-step process. Zein, which has affinity to both polar and non-polar groups, is expected to improve miscibility between the blends’ constituents and its overall biocompatibility. Mechanical properties of the blend were tested and further characterizations (Fourier transform infrared spectroscopy, thermal properties) were performed to analyze the effect of zein as an addition to the blend’s physical properties. The blend’s biocompatibility was examined by indirect methods (contact angle and weight gain after immersion in simulated body fluid) and in vitro analysis. No significant effect on the blend’s strength and stiffness was caused by adding zein. Hydrophilicity and cell affinity were improved when zein was added. Zein did not perform as a coupling agent that improved miscibility between polycaprolactone and starch, but its addition improved the blend’s biocompatibility.
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Affiliation(s)
- Fethma M Nor
- Department of Mechanical, Robotics, and Energy Engineering, Dongguk University, Korea
| | - Denni Kurniawan
- Department of Manufacturing and Industrial Engineering, Universiti Teknologi Malaysia, Malaysia
| | - Young-Kwon Seo
- Department of Medical Biotechnology, Dongguk University, Korea
| | - Jung-Keug Park
- Research Institute of Biotechnology, Dongguk University, Korea
- Department of Medical Biotechnology, Dongguk University, Korea
| | - Ho Yong Lee
- Department of Mechanical, Robotics, and Energy Engineering, Dongguk University, Korea
| | - Joong Yeon Lim
- Department of Mechanical, Robotics, and Energy Engineering, Dongguk University, Korea
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26
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Undale A, Fraser D, Hefferan T, Kopher RA, Herrick J, Evans GL, Li X, Kakar S, Hayes M, Atkinson E, Yaszemski MJ, Kaufman DS, Westendorf JJ, Khosla S. Induction of fracture repair by mesenchymal cells derived from human embryonic stem cells or bone marrow. J Orthop Res 2011; 29:1804-11. [PMID: 21674605 PMCID: PMC3179810 DOI: 10.1002/jor.21480] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/05/2011] [Accepted: 05/23/2011] [Indexed: 02/04/2023]
Abstract
Development of novel therapeutic approaches to repair fracture non-unions remains a critical clinical necessity. We evaluated the capacity of human embryonic stem cell (hESC)-derived mesenchymal stem/stromal cells (MSCs) to induce healing in a fracture non-union model in rats. In addition, we placed these findings in the context of parallel studies using human bone marrow MSCs (hBM-MSCs) or a no cell control group (n = 10-12 per group). Preliminary studies demonstrated that both for hESC-derived MSCs and hBM-MSCs, optimal induction of fracture healing required in vitro osteogenic differentiation of these cells. Based on biomechanical testing of fractured femurs, maximum torque, and stiffness were significantly greater in the hBM-MSC as compared to the control group that received no cells; values for these parameters in the hESC-derived MSC group were intermediate between the hBM-MSC and control groups, and not significantly different from the control group. However, some evidence of fracture healing was evident by X-ray in the hESC-derived MSC group. Our results thus indicate that while hESC-derived MSCs may have potential to induce fracture healing in non-unions, hBM-MSCs function more efficiently in this process. Additional studies are needed to further modify hESCs to achieve optimal fracture healing by these cells.
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Affiliation(s)
- Anita Undale
- Endocrine Research Unit, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Daniel Fraser
- Endocrine Research Unit, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Theresa Hefferan
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Ross A. Kopher
- Department of Medicine and Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
| | - James Herrick
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Glenda L. Evans
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Xiaodong Li
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Sanjeev Kakar
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Meredith Hayes
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | | | | | - Dan S. Kaufman
- Department of Medicine and Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
| | | | - Sundeep Khosla
- Endocrine Research Unit, College of Medicine, Mayo Clinic, Rochester, MN, USA
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The Role of Collagen Type I on Hematopoietic and Mesenchymal Stem Cells Expansion and Differentiation. ACTA ACUST UNITED AC 2011. [DOI: 10.4028/www.scientific.net/amr.409.111] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The three dimensional scaffold of the bone marrow (BM) niches is composed of various elements including extracellular matrix proteins and cell types, such as collagen type I (Col I) and stroma cells. Interaction of stem cells with their microenvironment is important for their regulation. In the marrow, Col I is mostly localized in the endosteal regions. The objective of this work was to investigate the role of Col I in the regulation of Hematopoietic Stem Cells (HSC) and Mesenchymal Stem Cells (MSC) growth. Col I was extracted from rat tail tendons and its purity confirmed. Human BM MSCs and umbilical cord blood (UCB) CD34+cells were used as Stem Cell sources. MSCs were cultured in medium with serum while CB CD34+cells were cultured without serum with cytokines. The impact of increasing concentrations of Col I (0-50 µg mL-1for coating) on the growth of Hematopoietic Progenitor Cells (HPC) and MSCs was investigated by cytometry, microscopy and clonogenic progenitor assays. Only a minority of CD34+cells expressed the Col I receptor α2β1prior to culture, while the opposite was observed when hematopoietic cells were placed in culture. Col I coated surfaces reduced the expansion of hematopoietic cells by 25% compared to control, while expansions of myeloid and MK progenitors were either unchanged or negatively affected by Col I, respectively. The differentiation of HPCs was also affected on Col I as demonstrated by differences in the frequencies of various cell lineages, such as CD34+cells, megakaryocytes (MK), erythrocytes and others. In contrast to HPCs, Col I surfaces increased MSCs proliferation but had little impact on osteoblasts derived from MSCs. Taken together, this study provides new insights into the regulatory activities of Col I on Stem Cells residing in the marrow.
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Schultheiss J, Seebach C, Henrich D, Wilhelm K, Barker JH, Frank J. Mesenchymal stem cell (MSC) and endothelial progenitor cell (EPC) growth and adhesion in six different bone graft substitutes. Eur J Trauma Emerg Surg 2011; 37:635-44. [DOI: 10.1007/s00068-011-0119-0] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2011] [Accepted: 05/16/2011] [Indexed: 10/18/2022]
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Angstmann M, Brinkmann I, Bieback K, Breitkreutz D, Maercker C. Monitoring human mesenchymal stromal cell differentiation by electrochemical impedance sensing. Cytotherapy 2011; 13:1074-89. [PMID: 21619493 DOI: 10.3109/14653249.2011.584863] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
BACKGROUND AIMS For their wide mesodermal differentiation potential, mesenchymal stromal/stem cells (MSC) are attractive candidates for tissue engineering. However, standardized quality control assays monitoring differentiation that are non-invasive and continuous over time are lacking. METHODS We employed a non-invasive assay, using two different systems, to discriminate osteogenic and adipogenic differentiation of MSC by monitoring impedance. Fibroblasts and keratinocytes served as non-specific controls. Impedance profiles were recorded comparing MSC from bone marrow and adipose tissue, either non-induced or induced for osteogenesis or adipogenesis, for 5-14 days, and correlated with differentiation markers assessed by reverse transcription-quantitative polymerase chain reaction and Western blot. Additionally, differentiation modulating effects of extracellular matrix components were analyzed. RESULTS Adhesion and growth-related impedance profiles of non-induced MSC roughly resembled those of fibroblasts, whereas keratinocytes differed significantly. Distinct from that, osteogenic induction of MSC revealed initially rapid and continuously rising impedance, corresponding to mineralized calcium matrix formation. Conversely, adipogenic induction caused shallower initial slopes and eventually declining profiles, corresponding to more compact, adipocyte-like cells with numerous lipid vacuoles. Pre-coating with either collagen type I or IV apparently favored osteogenesis and fibronectin adipogenesis. Impedance recordings correlated well with the extent of differentiation evaluated by histochemical staining and protein and gene expression. CONCLUSIONS Overall, our data demonstrate that impedance profiling offers a basis for standardized real-time, non-invasive high-throughput screening of MSC properties. It enables further testing of the influence of diffusible factors or extracellular matrix composites on MSC differentiation or maintenance of stemness, thus substantiating therapeutic application.
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Affiliation(s)
- Michael Angstmann
- Mannheim University of Applied Sciences, Biotechnology, Mannheim, Germany
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Copland IB, Galipeau J. Death and inflammation following somatic cell transplantation. Semin Immunopathol 2011; 33:535-50. [DOI: 10.1007/s00281-011-0274-8] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2010] [Accepted: 04/14/2011] [Indexed: 12/13/2022]
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