|
1
|
Ulpiano C, Salvador W, Franchi-Mendes T, Huang MC, Lin YH, Lin HT, Rodrigues CAV, Fernandes-Platzgummer A, Cabral JMS, Monteiro GA, da Silva CL. Continuous collection of human mesenchymal-stromal-cell-derived extracellular vesicles from a stirred tank reactor operated under xenogeneic-free conditions for therapeutic applications. Stem Cell Res Ther 2025; 16:210. [PMID: 40275409 PMCID: PMC12023423 DOI: 10.1186/s13287-025-04341-2] [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: 11/15/2024] [Accepted: 04/11/2025] [Indexed: 04/26/2025] Open
Abstract
BACKGROUND Mesenchymal-stromal-cell-derived extracellular vesicles (MSC-EVs) play a key role in the paracrine effects of MSC and have demonstrated therapeutic potential in various preclinical models. However, clinical translation is hindered by manufacturing practices relying on planar culture systems, fetal bovine serum (FBS)-supplemented media, and non-scalable, low-purity EV isolation methods that fail to meet dose and safety requirements, underscoring the need for innovative approaches. In this study, we developed a scalable platform to manufacture human MSC-EVs at clinically relevant numbers, integrating continuous collection of EV-enriched conditioned media (CM) using a stirred-tank reactor (STR) under xenogeneic-free conditions and a scalable downstream process. METHODS Wharton's jelly-derived MSC (MSC(WJ)) were expanded using microcarriers in a controlled STR using human platelet lysate (hPL)-supplemented medium. Then, a 3-day EV production stage, featuring continuous harvesting of the CM, was established using a novel serum-/xeno(geneic)-free exosome depleted-hPL supplement. For the isolation of MSC-EVs, a scalable process was implemented by pairing tangential flow filtration and anion exchange chromatography. Isolated MSC-EVs were characterised using nanoparticle tracking analysis, protein and zeta potential quantification, western blot analysis of EV protein markers, transmission electron microscopy and uptake studies of fluorescently labelled-EVs. RESULTS The system sustained the efficient expansion of MSC(WJ), reaching a total of (6.03 ± 0.181) x 107 cells after 7 days, which corresponds to a 30.1 ± 0.740-fold expansion. Upon a 3-day continuous CM harvesting, a total of (2.13 ± 0.301) x 1012 EVs were isolated corresponding to a particle yield factor of (1.26 ± 0.186) x 104 EVs/cell/day. MSC-EVs presented high purity levels ((5.53 ± 1.55) x 109 particles/µg), a homogeneous small size distribution (mean diameter of 115 ± 4.88 nm), a surface charge of -23.4 ± 6.23 mV, positive detection of tetraspanins CD9 and CD63 and syntenin-1 and displayed a typical cup-shaped morphology. MSC-EVs were readily incorporated by endothelial cells and two human breast cancer cell lines. CONCLUSIONS Overall, the scalable and Good Manufacturing Practices (GMP)-compliant platform established herein enabled the reproducible manufacturing of MSC-EVs with high purity and generally accepted characteristics concerning size, protein markers, surface charge, morphology, and cellular internalization, validating its potential for future clinical applications.
Collapse
Affiliation(s)
- Cristiana Ulpiano
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - William Salvador
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Teresa Franchi-Mendes
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | | | | | | | - Carlos A V Rodrigues
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Ana Fernandes-Platzgummer
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Joaquim M S Cabral
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Gabriel A Monteiro
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
| |
Collapse
|
|
2
|
Kang M, Yang Y, Zhang H, Zhang Y, Wu Y, Denslin V, Othman RB, Yang Z, Han J. Comparative Analysis of Serum and Serum-Free Medium Cultured Mesenchymal Stromal Cells for Cartilage Repair. Int J Mol Sci 2024; 25:10627. [PMID: 39408956 PMCID: PMC11476526 DOI: 10.3390/ijms251910627] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Revised: 09/23/2024] [Accepted: 09/28/2024] [Indexed: 10/20/2024] Open
Abstract
Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium's ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair.
Collapse
Affiliation(s)
- Meiqi Kang
- Critical Analytics for Manufacturing Personalised-Medicine (CAMP) Interdisciplinary Research Group (IRG), Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore 138602, Singapore; (M.K.); (Y.Y.); (R.B.O.)
| | - Yanmeng Yang
- Critical Analytics for Manufacturing Personalised-Medicine (CAMP) Interdisciplinary Research Group (IRG), Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore 138602, Singapore; (M.K.); (Y.Y.); (R.B.O.)
| | - Haifeng Zhang
- Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119288, Singapore; (H.Z.); (Y.Z.); (Y.W.); (V.D.)
- NUS Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore 117510, Singapore
| | - Yuan Zhang
- Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119288, Singapore; (H.Z.); (Y.Z.); (Y.W.); (V.D.)
- NUS Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore 117510, Singapore
| | - Yingnan Wu
- Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119288, Singapore; (H.Z.); (Y.Z.); (Y.W.); (V.D.)
- NUS Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore 117510, Singapore
| | - Vinitha Denslin
- Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119288, Singapore; (H.Z.); (Y.Z.); (Y.W.); (V.D.)
- NUS Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore 117510, Singapore
| | - Rashidah Binte Othman
- Critical Analytics for Manufacturing Personalised-Medicine (CAMP) Interdisciplinary Research Group (IRG), Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore 138602, Singapore; (M.K.); (Y.Y.); (R.B.O.)
| | - Zheng Yang
- Critical Analytics for Manufacturing Personalised-Medicine (CAMP) Interdisciplinary Research Group (IRG), Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore 138602, Singapore; (M.K.); (Y.Y.); (R.B.O.)
- Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119288, Singapore; (H.Z.); (Y.Z.); (Y.W.); (V.D.)
- NUS Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore 117510, Singapore
| | - Jongyoon Han
- Critical Analytics for Manufacturing Personalised-Medicine (CAMP) Interdisciplinary Research Group (IRG), Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore 138602, Singapore; (M.K.); (Y.Y.); (R.B.O.)
- Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
- Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
| |
Collapse
|
|
3
|
Patel AA, Mohamed AH, Rizaev J, Mallick AK, Qasim MT, Abdulmonem WA, Jamal A, Hattiwale HM, Kamal MA, Ahmad F. Application of mesenchymal stem cells derived from the umbilical cord or Wharton's jelly and their extracellular vesicles in the treatment of various diseases. Tissue Cell 2024; 89:102415. [PMID: 38851032 DOI: 10.1016/j.tice.2024.102415] [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: 02/13/2024] [Revised: 04/26/2024] [Accepted: 05/20/2024] [Indexed: 06/10/2024]
Abstract
Mesenchymal stem cells (MSCs) originating from the umbilical cord (UC) or Wharton's jelly (WJ) have attracted substantial interest due to their potential to augment therapeutic approaches for a wide range of disorders. These cells demonstrate a wide range of capabilities in the process of differentiating into a multitude of cell types. Additionally, they possess a significant capacity for proliferation and are conveniently accessible. Furthermore, they possess a status of being immune-privileged, exhibit minimal tumorigenic characteristics, and raise minimal ethical concerns. Consequently, they are well-suited candidates for tissue regeneration and the treatment of diseases. Additionally, UC-derived MSCs offer a substantial yield compared to other sources. The therapeutic effects of these MSCs are closely associated with the release of nanosized extracellular vesicles (EVs), including exosomes and microvesicles (MVs), containing lipids, microRNAs, and proteins that facilitate intercellular communication. Due to their reduced tumorigenic and immunogenic characteristics, in addition to their convenient manipulability, EVs have arisen as a viable alternative for the management of disorders. The favorable characteristics of UC-MSCs or WJ-MSCs and their EVs have generated significant attention in clinical investigations encompassing diverse pathologies. Therefore, we present a review encompassing current preclinical and clinical investigations, examining the implications of UC-MSCs in diverse diseases, including those affecting bone, cartilage, skin, liver, kidney, neural, lung, cardiovascular, muscle, and retinal tissues, as well as conditions like cancer, diabetes, sepsis, and others.
Collapse
Affiliation(s)
- Ayyub Ali Patel
- Clinical Biochemistry Department, College of Medicine, King Khalid University, Abha, Saudi Arabia
| | - Asma'a H Mohamed
- Biomedical Engineering Department, College of Engineering and Technologies, Al-Mustaqbal University, Hilla, Babil 51001, Iraq.
| | - Jasur Rizaev
- Department of Public Health and Healthcare management, Rector, Samarkand State Medical University, 18, Amir Temur Street, Samarkand, Uzbekistan
| | - Ayaz Khurram Mallick
- Clinical Biochemistry Department, College of Medicine, King Khalid University, Abha, Saudi Arabia
| | - Maytham T Qasim
- College of Health and Medical Technology, Al-Ayen University, Thi-Qar 64001, Iraq
| | - Waleed Al Abdulmonem
- Department of Pathology, College of Medicine, Qassim University, Buraidah, Saudi Arabia
| | - Azfar Jamal
- Department of Biology, College of Science Al-Zulfi, Majmaah University, Al-Majmaah 11952, Saudi Arabia; Health and Basic Science Research Centre, Majmaah University, Al-Majmaah 11952, Saudi Arabia
| | - Haroonrashid M Hattiwale
- Department of Basic Medical Sciences, College of Medicine, Majmaah University, Al-Majmaah 11952, Saudi Arabia.
| | - Mohammad Azhar Kamal
- Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Alkharj 11942, Saudi Arabia
| | - Fuzail Ahmad
- College of Applied Sciences, Almaarefa University, Diriya, Riyadh 13713, Saudi Arabia
| |
Collapse
|
|
4
|
Bandarra-Tavares H, Franchi-Mendes T, Ulpiano C, Morini S, Kaur N, Harris-Becker A, Vemuri MC, Cabral JMS, Fernandes-Platzgummer A, da Silva CL. Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system. Cytotherapy 2024; 26:749-756. [PMID: 38506771 DOI: 10.1016/j.jcyt.2024.03.001] [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: 07/26/2023] [Revised: 01/29/2024] [Accepted: 03/02/2024] [Indexed: 03/21/2024]
Abstract
BACKGROUND & AIMS Cell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium. METHODS MSC(WJ) isolated from three donors were cultured at a starting density of 1 × 106 cells per spinner flask, i.e., 2.8 × 103 cells per cm2 of dissolvable microcarrier surface area. After a 6-day expansion period of MSC(WJ), extracellular vesicles (EVs) were produced for 24 h. RESULTS Taking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1). CONCLUSIONS Taken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.
Collapse
Affiliation(s)
- Hélder Bandarra-Tavares
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Teresa Franchi-Mendes
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cristiana Ulpiano
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Sara Morini
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Navjot Kaur
- Cell and Gene Therapy, Thermo Fisher Scientific, Cell Biology, Frederick, Maryland, USA
| | - Abigail Harris-Becker
- Cell and Gene Therapy, Thermo Fisher Scientific, Cell Biology, Frederick, Maryland, USA
| | - Mohan C Vemuri
- Cell and Gene Therapy, Thermo Fisher Scientific, Cell Biology, Frederick, Maryland, USA
| | - Joaquim M S Cabral
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Ana Fernandes-Platzgummer
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
| |
Collapse
|
|
5
|
Rovere M, Reverberi D, Arnaldi P, Palamà MEF, Gentili C. Spheroid size influences cellular senescence and angiogenic potential of mesenchymal stromal cell-derived soluble factors and extracellular vesicles. Front Bioeng Biotechnol 2023; 11:1297644. [PMID: 38162179 PMCID: PMC10756914 DOI: 10.3389/fbioe.2023.1297644] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2023] [Accepted: 11/23/2023] [Indexed: 01/03/2024] Open
Abstract
Introduction: The secretome of mesenchymal stromal cells (MSCs) serves as an innovative tool employed in the regenerative medicine approach. In this particular context, three-dimensional (3D) culture systems are widely utilized to better replicate in vivo conditions and facilitate prolonged cell maintenance during culture. The use of spheroids enables the preservation of the classical phenotypical characteristics of MSCs. However, the distinct microenvironment within the spheroid may impact the secretome, thereby enhancing the angiogenic properties of adult MSCs that typically possess a reduced angiogenic potential compared to MSCs derived from perinatal tissues due to the hypoxia created in the internal region of the spheroid. Methods: In this study, large spheroids (2,600 cells, ∼300 μm diameter) and small spheroids (1,000 cells, ∼200 μm diameter) were used to examine the role of spheroid diameter in the generation of nutrients and oxygen gradients, cellular senescence, and the angiogenic potential of secreted factors and extracellular vesicles (EVs). Results: In this study, we demonstrate that large spheroids showed increased senescence and a secretome enriched in pro-angiogenic factors, as well as pro-inflammatory and anti-angiogenic cytokines, while small spheroids exhibited decreased senescence and a secretome enriched in pro-angiogenic molecules. We also demonstrated that 3D culture led to a higher secretion of EVs with classical phenotypic characteristics. Soluble factors and EVs from small spheroids exhibited higher angiogenic potential in a human umbilical vein endothelial cell (HUVEC) angiogenic assay. Discussion: These findings highlighted the necessity of choosing the appropriate culture system for obtaining soluble factors and EVs for specific therapeutic applications.
Collapse
Affiliation(s)
- Matteo Rovere
- Department of Experimental Medicine, University of Genoa, Genoa, Italy
| | | | - Pietro Arnaldi
- Department of Experimental Medicine, University of Genoa, Genoa, Italy
| | | | - Chiara Gentili
- Department of Experimental Medicine, University of Genoa, Genoa, Italy
| |
Collapse
|
|
6
|
Li CH, Zhao J, Zhang HY, Wang B. Banking of perinatal mesenchymal stem/stromal cells for stem cell-based personalized medicine over lifetime: Matters arising. World J Stem Cells 2023; 15:105-119. [PMID: 37181005 PMCID: PMC10173813 DOI: 10.4252/wjsc.v15.i4.105] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/06/2022] [Revised: 01/07/2023] [Accepted: 03/22/2023] [Indexed: 04/26/2023] Open
Abstract
Mesenchymal stromal/stem cells (MSCs) are currently applied in regenerative medicine and tissue engineering. Numerous clinical studies have indicated that MSCs from different tissue sources can provide therapeutic benefits for patients. MSCs derived from either human adult or perinatal tissues have their own unique advantages in their medical practices. Usually, clinical studies are conducted by using of cultured MSCs after thawing or short-term cryopreserved-then-thawed MSCs prior to administration for the treatment of a wide range of diseases and medical disorders. Currently, cryogenically banking perinatal MSCs for potential personalized medicine for later use in lifetime has raised growing interest in China as well as in many other countries. Meanwhile, this has led to questions regarding the availability, stability, consistency, multipotency, and therapeutic efficiency of the potential perinatal MSC-derived therapeutic products after long-term cryostorage. This opinion review does not minimize any therapeutic benefit of perinatal MSCs in many diseases after short-term cryopreservation. This article mainly describes what is known about banking perinatal MSCs in China and, importantly, it is to recognize the limitation and uncertainty of the perinatal MSCs stored in cryobanks for stem cell medical treatments in whole life. This article also provides several recommendations for banking of perinatal MSCs for potentially future personalized medicine, albeit it is impossible to anticipate whether the donor will benefit from banked MSCs during her/his lifetime.
Collapse
Affiliation(s)
- Cheng-Hai Li
- Stem Cell Program of Clinical Research Center, People's Hospital of Zhengzhou University and Henan Provincial People's Hospital, Zhengzhou 450003, Henan Province, China
| | - Jing Zhao
- Department of Clinical Laboratory, People's Hospital of Zhengzhou University and Henan Provincial People's Hospital, Zhengzhou 450003, Henan Province, China
| | - Hong-Yan Zhang
- Department of Pharmacy, Fuwai Central China Cardiovascular Hospital, Zhengzhou 450000, Henan Province, China
| | - Bin Wang
- Department of Neurosurgery, People's Hospital of Zhengzhou University and Henan Provincial People's Hospital, Zhengzhou 450003, Henan Province, China.
| |
Collapse
|
|
7
|
Marques CR, Fuzeta MDA, Dos Santos Cunha RM, Pereira-Sousa J, Silva D, Campos J, Teixeira-Castro A, Sousa RA, Fernandes-Platzgummer A, da Silva CL, Salgado AJ. Neurodifferentiation and Neuroprotection Potential of Mesenchymal Stromal Cell-Derived Secretome Produced in Different Dynamic Systems. Biomedicines 2023; 11:biomedicines11051240. [PMID: 37238911 DOI: 10.3390/biomedicines11051240] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 03/30/2023] [Accepted: 04/14/2023] [Indexed: 05/28/2023] Open
Abstract
Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by the degeneration of the dopamine (DA) neurons in the substantia nigra pars compacta, leading to a loss of DA in the basal ganglia. The presence of aggregates of alpha-synuclein (α-synuclein) is seen as the main contributor to the pathogenesis and progression of PD. Evidence suggests that the secretome of mesenchymal stromal cells (MSC) could be a potential cell-free therapy for PD. However, to accelerate the integration of this therapy in the clinical setting, there is still the need to develop a protocol for the large-scale production of secretome under good manufacturing practices (GMP) guidelines. Bioreactors have the capacity to produce large quantities of secretomes in a scalable manner, surpassing the limitations of planar static culture systems. However, few studies focused on the influence of the culture system used to expand MSC, on the secretome composition. In this work, we studied the capacity of the secretome produced by bone marrow-derived mesenchymal stromal cells (BMSC) expanded in a spinner flask (SP) and in a Vertical-Wheel™ bioreactor (VWBR) system, to induce neurodifferentiation of human neural progenitor cells (hNPCs) and to prevent dopaminergic neuron degeneration caused by the overexpression of α-synuclein in one Caenorhabditis elegans model of PD. Results showed that secretomes from both systems were able to induce neurodifferentiation, though the secretome produced in the SP system had a greater effect. Additionally, in the conditions of our study, only the secretome produced in SP had a neuroprotective potential. Lastly, the secretomes had different profiles regarding the presence and/or specific intensity of different molecules, namely, interleukin (IL)-6, IL-4, matrix metalloproteinase-2 (MMP2), and 3 (MMP3), tumor necrosis factor-beta (TNF-β), osteopontin, nerve growth factor beta (NGFβ), granulocyte colony-stimulating factor (GCSF), heparin-binding (HB) epithelial growth factor (EGF)-like growth factor (HB-EGF), and IL-13. Overall, our results suggest that the culture conditions might have influenced the secretory profiles of cultured cells and, consequently, the observed effects. Additional studies should further explore the effects that different culture systems have on the secretome potential of PD.
Collapse
Affiliation(s)
- Cláudia Raquel Marques
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal
- ICVS-3Bs PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal
| | - Miguel de Almeida Fuzeta
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
- Associate Laboratory i4HB-Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
| | - Raquel Medina Dos Santos Cunha
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
- Associate Laboratory i4HB-Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
| | - Joana Pereira-Sousa
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal
- ICVS-3Bs PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal
| | - Deolinda Silva
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal
- ICVS-3Bs PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal
| | - Jonas Campos
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal
- ICVS-3Bs PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal
| | - Andreia Teixeira-Castro
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal
- ICVS-3Bs PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal
| | - Rui Amandi Sousa
- Stemmatters, Biotecnologia e Medicina Regenerativa S.A., 4805-017 Barco, Portugal
| | - Ana Fernandes-Platzgummer
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
- Associate Laboratory i4HB-Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
- Associate Laboratory i4HB-Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
| | - António José Salgado
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal
- ICVS-3Bs PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal
| |
Collapse
|
|
8
|
Zanicotti DG, Milne TJ, Coates DE. Culturing Adipose-Derived Stem Cells Under Serum-Free Conditions. Methods Mol Biol 2023; 2588:407-415. [PMID: 36418700 DOI: 10.1007/978-1-0716-2780-8_23] [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] [Indexed: 06/16/2023]
Abstract
Growing adipose-derived stem cells (ADSC) in serum-free conditions is important as it represents a way of expanding multipotent cells in a clinical grade medium. Most cultured ADSC are expanded and tested in serum-containing media, which can pose significant health risks if these cells were used in clinical applications. Moreover, cells grown in serum-free conditions behave very different than those cultured in serum-containing media. Here, we present a technique to culture adipose-derived stem cells in serum-free conditions. The methods described in this chapter were optimized for ovine ADSC. The appropriate optimization should be done for other cell lines.
Collapse
Affiliation(s)
- Diogo Godoy Zanicotti
- Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand
| | - Trudy J Milne
- Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand
| | - Dawn E Coates
- Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand.
| |
Collapse
|
|
9
|
Tan L, Liu X, Dou H, Hou Y. Characteristics and regulation of mesenchymal stem cell plasticity by the microenvironment — specific factors involved in the regulation of MSC plasticity. Genes Dis 2022; 9:296-309. [PMID: 35224147 PMCID: PMC8843883 DOI: 10.1016/j.gendis.2020.10.006] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2020] [Revised: 10/05/2020] [Accepted: 10/22/2020] [Indexed: 02/07/2023] Open
Abstract
Mesenchymal stem cells (MSCs), multipotent stromal cells, have attracted extensive attention in the field of regenerative medicine and cell therapy due to the capacity of self-renewal, multilineage differentiation, and immune regulation. MSCs have different cellular effects in different diseases, and even have markedly different curative effects with different tissue sources, indicating the plasticity of MSCs. The phenotypes, secreted factors, and proliferative, migratory, differentiating, and immunomodulatory effects of MSCs depend on certain mediators present in their microenvironment. Understanding microenvironmental factors and their internal mechanisms in MSC responses may help in subsequent prediction and improvement of clinical benefits. This review highlighted the recent advances in MSC plasticity in the physiological and pathological microenvironment and multiple microenvironmental factors regulating MSC plasticity. It also highlighted some progress in the underlying molecular mechanisms of MSC remodeling in the microenvironment. It might provide references for the improvement in vitro culture of MSCs, clinical application, and in vivo induction.
Collapse
|
|
10
|
Silicon-Gold Nanoparticles Affect Wharton's Jelly Phenotype and Secretome during Tri-Lineage Differentiation. Int J Mol Sci 2022; 23:ijms23042134. [PMID: 35216249 PMCID: PMC8874983 DOI: 10.3390/ijms23042134] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2021] [Revised: 02/10/2022] [Accepted: 02/11/2022] [Indexed: 12/14/2022] Open
Abstract
Multiple studies have demonstrated that various nanoparticles (NPs) stimulate osteogenic differentiation of mesenchymal stem cells (MSCs) and inhibit adipogenic ones. The mechanisms of these effects are not determined. The aim of this paper was to estimate Wharton’s Jelly MSCs phenotype and humoral factor production during tri-lineage differentiation per se and in the presence of silicon–gold NPs. Silicon (SiNPs), gold (AuNPs), and 10% Au-doped Si nanoparticles (SiAuNPs) were synthesized by laser ablation, characterized, and studied in MSC cultures before and during differentiation. Humoral factor production (n = 41) was analyzed by Luminex technology. NPs were nontoxic, did not induce ROS production, and stimulated G-CSF, GM-CSF, VEGF, CXCL1 (GRO) production in four day MSC cultures. During MSC differentiation, all NPs stimulated CD13 and CD90 expression in osteogenic cultures. MSC differentiation resulted in a decrease in multiple humoral factor production to day 14 of incubation. NPs did not significantly affect the production in chondrogenic cultures and stimulated it in both osteogenic and adipogenic ones. The major difference in the protein production between osteogenic and adipogenic MSC cultures in the presence of NPs was VEGF level, which was unaffected in osteogenic cells and 4–9 times increased in adipogenic ones. The effects of NPs decreased in a row AuNPs > SiAuNPs > SiNPs. Taken collectively, high expression of CD13 and CD90 by MSCs and critical level of VEGF production can, at least, partially explain the stimulatory effect of NPs on MSC osteogenic differentiation.
Collapse
|
|
11
|
Petry F, Salzig D. Impact of Bioreactor Geometry on Mesenchymal Stem Cell Production in Stirred‐Tank Bioreactors. CHEM-ING-TECH 2021. [DOI: 10.1002/cite.202100041] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Affiliation(s)
- Florian Petry
- University of Applied Sciences Mittelhessen Institute of Bioprocess Engineering and Pharmaceutical Technology Wiesenstraße 14 35390 Giessen Germany
| | - Denise Salzig
- University of Applied Sciences Mittelhessen Institute of Bioprocess Engineering and Pharmaceutical Technology Wiesenstraße 14 35390 Giessen Germany
| |
Collapse
|
|
12
|
Rizzo MI, Tomao L, Tedesco S, Cajozzo M, Esposito M, De Stefanis C, Ferranti AM, Mezzogori D, Palmieri A, Pozzato G, Algeri M, Locatelli F, Leone L, Zama M. Engineered mucoperiosteal scaffold for cleft palate regeneration towards the non-immunogenic transplantation. Sci Rep 2021; 11:14570. [PMID: 34272436 PMCID: PMC8285425 DOI: 10.1038/s41598-021-93951-w] [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: 11/28/2020] [Accepted: 05/25/2021] [Indexed: 12/15/2022] Open
Abstract
Cleft lip and palate (CL/P) is the most prevalent craniofacial birth defect in humans. None of the surgical procedures currently used for CL/P repair lead to definitive correction of hard palate bone interruption. Advances in tissue engineering and regenerative medicine aim to develop new strategies to restore palatal bone interruption by using tissue or organ-decellularized bioscaffolds seeded with host cells. Aim of this study was to set up a new natural scaffold deriving from a decellularized porcine mucoperiosteum, engineered by an innovative micro-perforation procedure based on Quantum Molecular Resonance (QMR) and then subjected to in vitro recellularization with human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Our results demonstrated the efficiency of decellularization treatment gaining a natural, non-immunogenic scaffold with preserved collagen microenvironment that displays a favorable support to hMSC engraftment, spreading and differentiation. Ultrastructural analysis showed that the micro-perforation procedure preserved the collagen mesh, increasing the osteoinductive potential for mesenchymal precursor cells. In conclusion, we developed a novel tissue engineering protocol to obtain a non-immunogenic mucoperiosteal scaffold suitable for allogenic transplantation and CL/P repair. The innovative micro-perforation procedure improving hMSC osteogenic differentiation potentially impacts for enhanced palatal bone regeneration leading to future clinical applications in humans.
Collapse
Affiliation(s)
- M I Rizzo
- Plastic and Maxillofacial Surgery Department, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - L Tomao
- Department of Pediatric Onco-Hematology and Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - S Tedesco
- Telea Biotech e Telea Electronic Engineering, Sandrigo, VI, Italy
| | - M Cajozzo
- Plastic and Maxillofacial Surgery Department, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - M Esposito
- Plastic and Maxillofacial Surgery Department, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - C De Stefanis
- Research Laboratories, Histology Core Facility, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - A M Ferranti
- Department of Neuroscience, Università Cattolica del Sacro Cuore, 00168, Rome, Italy
| | - D Mezzogori
- Department of Neuroscience, Università Cattolica del Sacro Cuore, 00168, Rome, Italy
| | - A Palmieri
- Department of Cardiovascular, Endocrine-Metabolic Diseases and Aging, National Institute of Health, Rome, Italy
| | - G Pozzato
- Telea Biotech e Telea Electronic Engineering, Sandrigo, VI, Italy
| | - M Algeri
- Department of Pediatric Onco-Hematology and Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| | - F Locatelli
- Department of Pediatric Onco-Hematology and Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.,Department of Gynecology/Obstetrics & Pediatrics, Sapienza University of Rome, Rome, Italy
| | - L Leone
- Department of Neuroscience, Università Cattolica del Sacro Cuore, 00168, Rome, Italy. .,Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168, Rome, Italy.
| | - M Zama
- Plastic and Maxillofacial Surgery Department, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
| |
Collapse
|
|
13
|
Bucar S, Branco ADDM, Mata MF, Milhano JC, Caramalho Í, Cabral JMS, Fernandes-Platzgummer A, da Silva CL. Influence of the mesenchymal stromal cell source on the hematopoietic supportive capacity of umbilical cord blood-derived CD34 +-enriched cells. Stem Cell Res Ther 2021; 12:399. [PMID: 34256848 PMCID: PMC8278708 DOI: 10.1186/s13287-021-02474-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Accepted: 06/24/2021] [Indexed: 12/18/2022] Open
Abstract
Background Umbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem/progenitor cells (HSPC). To overcome the low cell number per UCB unit, ex vivo expansion of UCB HSPC in co-culture with mesenchymal stromal cells (MSC) has been established. Bone marrow (BM)-derived MSC have been the standard choice, but the use of MSC from alternative sources, less invasive and discardable, could ease clinical translation of an expanded CD34+ cell product. Here, we compare the capacity of BM-, umbilical cord matrix (UCM)-, and adipose tissue (AT)-derived MSC, expanded with/without xenogeneic components, to expand/maintain UCB CD34+-enriched cells ex vivo. Methods UCB CD34+-enriched cells were isolated from cryopreserved mononuclear cells and cultured for 7 days over an established feeder layer (FL) of BM-, UCM-, or AT-derived MSC, previously expanded using fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (HPL) supplemented medium. UCB cells were cultured in serum-free medium supplemented with SCF/TPO/FLT3-L/bFGF. Fold increase in total nucleated cells (TNC) as well as immunophenotype and clonogenic potential (cobblestone area-forming cells and colony-forming unit assays) of the expanded hematopoietic cells were assessed. Results MSC from all sources effectively supported UCB HSPC expansion/maintenance ex vivo, with expansion factors (in TNC) superior to 50x, 70x, and 80x in UCM-, BM-, and AT-derived MSC co-cultures, respectively. Specifically, AT-derived MSC co-culture resulted in expanded cells with similar phenotypic profile compared to BM-derived MSC, but resulting in higher total cell numbers. Importantly, a subpopulation of more primitive cells (CD34+CD90+) was maintained in all co-cultures. In addition, the presence of a MSC FL was essential to maintain and expand a subpopulation of progenitor T cells (CD34+CD7+). The use of HPL to expand MSC prior to co-culture establishment did not influence the expansion potential of UCB cells. Conclusions AT represents a promising alternative to BM as a source of MSC for co-culture protocols to expand/maintain HSPC ex vivo. On the other hand, UCM-derived MSC demonstrated inferior hematopoietic supportive capacity compared to MSC from adult tissues. Despite HPL being considered an alternative to FBS for clinical-scale manufacturing of MSC, further studies are needed to determine its impact on the hematopoietic supportive capacity of these cells.
Collapse
Affiliation(s)
- Sara Bucar
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,Associate Laboratory i4HB - Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - André Dargen de Matos Branco
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,Associate Laboratory i4HB - Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Márcia F Mata
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - João Coutinho Milhano
- Hospital São Francisco Xavier, Centro Hospitalar de Lisboa Ocidental, Lisboa, Portugal
| | | | - Joaquim M S Cabral
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,Associate Laboratory i4HB - Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Ana Fernandes-Platzgummer
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,Associate Laboratory i4HB - Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal. .,Associate Laboratory i4HB - Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
| |
Collapse
|
|
14
|
Serra J, Alves CPA, Cabral JMS, Monteiro GA, da Silva CL, Prazeres DMF. Minicircle-based expression of vascular endothelial growth factor in mesenchymal stromal cells from diverse human tissues. J Gene Med 2021; 23:e3342. [PMID: 33870576 DOI: 10.1002/jgm.3342] [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: 01/29/2021] [Revised: 03/26/2021] [Accepted: 04/10/2021] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND Mesenchymal stromal cells (MSC) have been exploited for the treatment of ischemic diseases given their angiogenic potential. Despite bone marrow (BM) being the most studied tissue source, cells with similar intrinsic properties can be isolated from adipose tissue (AT) and umbilical cord matrix (UCM). The present study aims to compare the angiogenic potential of MSC obtained from BM, AT and UCM that were genetically modified with vascular endothelial growth factor (VEGF)-encoding minicircle (MC) vectors. The overexpression of VEGF combined with the intrinsic properties of MSC could represent a promising strategy towards angiogenic therapies. METHODS We established a microporation-based protocol to transfect human MSC using VEGF-encoding MC (MC-VEGF). VEGF production levels were measured by an enzyme-linked immunosorbent assay and a quantitative polymerase chain reaction. The in vitro angiogenic potential of transfected cells was quantified using cell tube formation and migration functional studies. RESULTS MSC isolated from BM, AT or UCM showed similar levels of VEGF secretion after transfection with MC-VEGF. Those values were significantly higher when compared to non-transfected cells, indicating an effective enhancement of VEGF production. Transfected cells displayed higher in vitro angiogenic potential than non-transfected controls, as demonstrated by functional in vitro assays. No significant differences were observed among cells from different sources. CONCLUSIONS Minicircles can be successfully used to transiently overexpress VEGF in human MSC, regardless of the cell tissue source, representing an important advantage in a clinical context (i.e., angiogenic therapy) because a standard protocol might be applied to MSC of different tissue sources, which can be differentially selected according to the application (e.g., autologous versus allogeneic settings).
Collapse
Affiliation(s)
- Joana Serra
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia P A Alves
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Joaquim M S Cabral
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Gabriel A Monteiro
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Duarte Miguel F Prazeres
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| |
Collapse
|
|
15
|
Noronha NC, Mizukami A, Orellana MD, Oliveira MC, Covas DT, Swiech K, Malmegrim KC. Hypoxia priming improves in vitro angiogenic properties of umbilical cord derived-mesenchymal stromal cells expanded in stirred-tank bioreactor. Biochem Eng J 2021. [DOI: 10.1016/j.bej.2021.107949] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
|
|
16
|
Tynecka M, Moniuszko M, Eljaszewicz A. Old Friends with Unexploited Perspectives: Current Advances in Mesenchymal Stem Cell-Based Therapies in Asthma. Stem Cell Rev Rep 2021; 17:1323-1342. [PMID: 33649900 PMCID: PMC7919631 DOI: 10.1007/s12015-021-10137-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/10/2021] [Indexed: 02/07/2023]
Abstract
Mesenchymal stem cells (MSCs) have a great regenerative and immunomodulatory potential that was successfully tested in numerous pre-clinical and clinical studies of various degenerative, hematological and inflammatory disorders. Over the last few decades, substantial immunoregulatory effects of MSC treatment were widely observed in different experimental models of asthma. Therefore, it is tempting to speculate that stem cell-based treatment could become an attractive means to better suppress asthmatic airway inflammation, especially in subjects resistant to currently available anti-inflammatory therapies. In this review, we discuss mechanisms accounting for potent immunosuppressive properties of MSCs and the rationale for their use in asthma. We describe in detail an intriguing interplay between MSCs and other crucial players in the immune system as well as lung microenvironment. Finally, we reveal the potential of MSCs in maintaining airway epithelial integrity and alleviating lung remodeling.
Collapse
Affiliation(s)
- Marlena Tynecka
- Department of Regenerative Medicine and Immune Regulation, Medical University of Bialystok, ul. Waszyngtona 13, 15-269, Białystok, Poland
| | - Marcin Moniuszko
- Department of Regenerative Medicine and Immune Regulation, Medical University of Bialystok, ul. Waszyngtona 13, 15-269, Białystok, Poland.
- Department of Allergology and Internal Medicine, Medical University of Bialystok, ul. M. Skłodowskiej-Curie 24A, Białystok, 15-276, Poland.
| | - Andrzej Eljaszewicz
- Department of Regenerative Medicine and Immune Regulation, Medical University of Bialystok, ul. Waszyngtona 13, 15-269, Białystok, Poland.
| |
Collapse
|
|
17
|
Santos GC, Silva DN, Fortuna V, Silveira BM, Orge ID, de Santana TA, Sampaio GL, Paredes BD, Ribeiro-Dos-Santos R, Soares MBP. Leukemia Inhibitory Factor (LIF) Overexpression Increases the Angiogenic Potential of Bone Marrow Mesenchymal Stem/Stromal Cells. Front Cell Dev Biol 2020; 8:778. [PMID: 32923442 PMCID: PMC7456813 DOI: 10.3389/fcell.2020.00778] [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: 02/28/2020] [Accepted: 07/24/2020] [Indexed: 12/15/2022] Open
Abstract
Mesenchymal stem/stromal cells (MSCs) have the ability to secrete bioactive molecules, exerting multiple biological effects, such as tissue regeneration, reduction of inflammation, and neovascularization. The therapeutic potential of MSCs can be increased by genetic modification to overexpress cytokines and growth factors. Here we produced mouse MSCs overexpressing human leukemia inhibitory factor (LIF) to assess their proangiogenic potential in vitro and in vivo. Mouse bone marrow-derived MSCs were transduced by using a second-generation lentiviral system to express human LIF. Leukemia inhibitory factor expression was confirmed by RT-qPCR and by ELISA, allowing the quantification of the transcript and secreted protein, respectively. Flow cytometry analysis and trilineage differentiation assay showed that the MSC_LIF cell line maintained the immunophenotype and a multipotency characteristic of MSCs. The immunosuppressive activity of MSC_LIF was confirmed using a lymphoproliferation assay. Moreover, gene expression analysis demonstrated upregulation of genes coding for strategic factors in the neovascularization process, such as angiogenin, IL-8, MCP-1, and VEGF, and for the perivascular cell markers αSMA, Col4a1, SM22, and NG2. To evaluate the pro-angiogenic potential of MSC_LIF, we first tested its effects on endothelial cells obtained from umbilical vein in a scratch wound healing assay. Conditioned medium (CM) from MSC_LIF promoted a significant increase in cell migration compared to CM from control MSC. Additionally, in vitro tube formation of endothelial cells was increased by the presence of MSC_LIF, as shown in microvessel sprouting in aortic ring cultures. Finally, an in vivo Matrigel plug assay was performed, showing that MSC_LIF were more potent in promoting in vivo angiogenesis and tissue vascularization than control MSCs. In conclusion, LIF overexpression is a promising strategy to increase the proangiogenic potential of MSCs and sets precedents for future investigations of their potential applications for the treatment of ischemic diseases and tissue repair.
Collapse
Affiliation(s)
- Girlaine Café Santos
- Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Brazil.,Health Institute of Technology, SENAI-CIMATEC, Salvador, Brazil
| | - Daniela Nascimento Silva
- Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Brazil.,Health Institute of Technology, SENAI-CIMATEC, Salvador, Brazil
| | - Vitor Fortuna
- Health Sciences Institute, Federal University of Bahia, Salvador, Brazil
| | | | - Iasmim Diniz Orge
- Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Brazil.,Health Institute of Technology, SENAI-CIMATEC, Salvador, Brazil
| | - Thaís Alves de Santana
- Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Brazil.,Health Institute of Technology, SENAI-CIMATEC, Salvador, Brazil
| | | | | | - Ricardo Ribeiro-Dos-Santos
- Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Brazil.,Health Institute of Technology, SENAI-CIMATEC, Salvador, Brazil.,National Institute of Science and Technology for Regenerative Medicine, Rio de Janeiro, Brazil
| | - Milena Botelho Pereira Soares
- Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Brazil.,Health Institute of Technology, SENAI-CIMATEC, Salvador, Brazil.,National Institute of Science and Technology for Regenerative Medicine, Rio de Janeiro, Brazil
| |
Collapse
|