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Carrillo Sanchez B, Hinchliffe M, Ellis M, Simpson C, Humphreys D, Sweeney B, Bracewell DG. Chinese hamster ovary cell line engineering strategies for modular production of custom extracellular vesicles. Biotechnol Bioeng 2024; 121:2907-2923. [PMID: 38924052 DOI: 10.1002/bit.28776] [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: 03/11/2024] [Revised: 05/23/2024] [Accepted: 06/09/2024] [Indexed: 06/28/2024]
Abstract
Continuously secreted by all cell types, extracellular vesicles (EVs) are small membrane-bound structures which shuttle bioactive cargo between cells across their external environment. Their central role as natural molecular messengers and ability to cross biological barriers has garnered significant attention in the use of EVs as therapeutic delivery vehicles. Still, harnessing the potential of EVs is faced with many obstacles. A cell line engineering approach can be used to exploit EVs to encapsulate a bespoke cargo of interest. However, full details regarding native EV-loading mechanisms remain under debate, making this a challenge. While Chinese hamster ovary (CHO) cells are well known to be the preferred host for recombinant therapeutic protein production, their application as an EV producer cell host has been largely overlooked. In this study, we engineered CHO DG44 cells to produce custom EVs with bespoke cargo. To this end, genetic constructs employing split green fluorescent protein technology were designed for tagging both CD81 and protein cargoes to enable EV loading via self-assembling activity. To demonstrate this, NanoLuc and mCherry were used as model reporter cargoes to validate engineered loading into EVs. Experimental findings indicated that our custom EV approach produced vesicles with up to 15-fold greater cargo compared with commonly used passive loading strategies. When applied to recipient cells, we observed a dose-dependent increase in cargo activity, suggesting successful delivery of engineered cargo via our custom CHO EVs.
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Affiliation(s)
- Braulio Carrillo Sanchez
- Department of Biochemical Engineering, University College London, London, UK
- UCB Pharma, Slough, Berkshire, UK
| | | | | | | | | | | | - Daniel G Bracewell
- Department of Biochemical Engineering, University College London, London, UK
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2
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Bobis-Wozowicz S, Paw M, Sarna M, Kędracka-Krok S, Nit K, Błażowska N, Dobosz A, Hammad R, Cathomen T, Zuba-Surma E, Tyszka-Czochara M, Madeja Z. Hypoxic extracellular vesicles from hiPSCs protect cardiomyocytes from oxidative damage by transferring antioxidant proteins and enhancing Akt/Erk/NRF2 signaling. Cell Commun Signal 2024; 22:356. [PMID: 38982464 PMCID: PMC11232324 DOI: 10.1186/s12964-024-01722-7] [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: 02/25/2024] [Accepted: 06/21/2024] [Indexed: 07/11/2024] Open
Abstract
BACKGROUND Stem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection. METHODS EVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385. RESULTS We demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs. CONCLUSIONS In this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.
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Affiliation(s)
- Sylwia Bobis-Wozowicz
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland.
| | - Milena Paw
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
| | - Michał Sarna
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Biophysics, Jagiellonian University, Krakow, Poland
| | - Sylwia Kędracka-Krok
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Physical Biochemistry, Jagiellonian University, Krakow, Poland
| | - Kinga Nit
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
| | - Natalia Błażowska
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
| | - Anna Dobosz
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
| | - Ruba Hammad
- Freiburg iPS Core Facility, Institute for Transfusion Medicine and Gene Therapy, Medical Center- University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), University of Freiburg, Freiburg, Germany
| | - Toni Cathomen
- Freiburg iPS Core Facility, Institute for Transfusion Medicine and Gene Therapy, Medical Center- University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), University of Freiburg, Freiburg, Germany
| | - Ewa Zuba-Surma
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
| | - Małgorzata Tyszka-Czochara
- Faculty of Pharmacy, Department of Food Chemistry and Nutrition, Jagiellonian University Medical College, Kraków, Poland
| | - Zbigniew Madeja
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
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Li J, Lu Z, Xu L, Wang J, Qian S, Hu Q, Ge Y. Poly(ethylenimine)-Cyclodextrin-Based Cationic Polymer Mediated HIF-1α Gene Delivery for Hindlimb Ischemia Treatment. ACS APPLIED BIO MATERIALS 2024; 7:1081-1094. [PMID: 38294873 DOI: 10.1021/acsabm.3c01020] [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: 02/01/2024]
Abstract
Hindlimb ischemia is a common disease worldwide featured by the sudden decrease in limb perfusion, which usually causes a potential threat to limb viability and even amputation or death. Revascularization has been defined as the gold-standard therapy for hindlimb ischemia. Considering that vascular injury recovery requires cellular adaptation to the hypoxia, hypoxia-inducible factor 1 α (HIF-1α) is a potential gene for tissue restoration and angiogenesis. In this manuscript, effective gene delivery vector PEI-β-CD (PC) was reported for the first application in the hindlimb ischemia treatment to deliver HIF-1α plasmid in vitro and in vivo. Our in vitro finding demonstrated that PC/HIF-1α-pDNA could be successfully entered into the cells and mediated efficient gene transfection with good biocompatibility. More importantly, under hypoxic conditions, PC/HIF-1α-pDNA could up-regulate the HUEVC cell viability. In addition, the mRNA levels of VEGF, Ang-1, and PDGF were upregulated, and transcriptome results also demonstrated that the cell-related function of response to hypoxia was enhanced. The therapeutic effect of PC/HIF-1α-pDNA was further estimated in a murine acute hindlimb ischemia model, which demonstrated that intramuscular injection of PC/HIF-1α-pDNA resulted in significantly increased blood perfusion and alleviation in tissue damage, such as tissue fibrosis and inflammation. The results provide a rationale that HIF-1α-mediated gene therapy might be a practical strategy for the treatment of limb ischemia.
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Affiliation(s)
- Jingyu Li
- College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China
| | - Zhuoting Lu
- College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China
| | - Liwang Xu
- College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China
| | - Jing Wang
- Cancer Center, Department of Ultrasound Medicine, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou 314408, China
| | - Shaojie Qian
- Center for Rehabilitation Medicine, Department of Anesthesiology, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou 314408, China
| | - Qinglian Hu
- College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China
| | - Yunfen Ge
- Center for Rehabilitation Medicine, Department of Anesthesiology, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou 314408, China
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Muok L, Sun L, Esmonde C, Worden H, Vied C, Duke L, Ma S, Zeng O, Driscoll T, Jung S, Li Y. Extracellular vesicle biogenesis of three-dimensional human pluripotent stem cells in a novel Vertical-Wheel bioreactor. JOURNAL OF EXTRACELLULAR BIOLOGY 2024; 3:e133. [PMID: 38938678 PMCID: PMC11080838 DOI: 10.1002/jex2.133] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Revised: 12/01/2023] [Accepted: 12/18/2023] [Indexed: 06/29/2024]
Abstract
Extracellular vesicles (EVs) secreted by human-induced pluripotent stem cells (hiPSCs) have great potential as cell-free therapies in various diseases, including prevention of blood-brain barrier senescence and stroke. However, there are still challenges in pre-clinical and clinical use of hiPSC-EVs due to the need for large-scale production of a large quantity. Vertical-Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC-EVs using a scalable aggregate or microcarrier-based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3-D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA-seq, respectively. The in vitro functional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3-D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA-seq. The microcarrier cultures had at least 17-23 fold higher EV secretion, and EV collection in mTeSR had 2.7-3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA-seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt-related pathways). hiPSC-EVs demonstrated the ability of stimulating proliferation and M2 polarization of microglia in vitro. HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti-aging study.
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Affiliation(s)
- Laureana Muok
- Department of Chemical and Biomedical Engineering, FAMU‐FSU College of EngineeringFlorida State UniversityTallahasseeFloridaUSA
| | - Li Sun
- Department of Chemical and Biomedical Engineering, FAMU‐FSU College of EngineeringFlorida State UniversityTallahasseeFloridaUSA
- Department of Biomedical Sciences, College of MedicineFlorida State UniversityTallahasseeFloridaUSA
| | - Colin Esmonde
- Department of Chemical and Biomedical Engineering, FAMU‐FSU College of EngineeringFlorida State UniversityTallahasseeFloridaUSA
| | | | - Cynthia Vied
- Department of Biomedical Sciences, College of MedicineFlorida State UniversityTallahasseeFloridaUSA
| | - Leanne Duke
- Department of Biomedical Sciences, College of MedicineFlorida State UniversityTallahasseeFloridaUSA
| | - Shaoyang Ma
- Department of Chemical and Biomedical Engineering, FAMU‐FSU College of EngineeringFlorida State UniversityTallahasseeFloridaUSA
| | - Olivia Zeng
- Department of Chemical and Biomedical Engineering, FAMU‐FSU College of EngineeringFlorida State UniversityTallahasseeFloridaUSA
| | - Tristan Driscoll
- Department of Chemical and Biomedical Engineering, FAMU‐FSU College of EngineeringFlorida State UniversityTallahasseeFloridaUSA
| | | | - Yan Li
- Department of Chemical and Biomedical Engineering, FAMU‐FSU College of EngineeringFlorida State UniversityTallahasseeFloridaUSA
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Paw M, Kusiak AA, Nit K, Litewka JJ, Piejko M, Wnuk D, Sarna M, Fic K, Stopa KB, Hammad R, Barczyk-Woznicka O, Cathomen T, Zuba-Surma E, Madeja Z, Ferdek PE, Bobis-Wozowicz S. Hypoxia enhances anti-fibrotic properties of extracellular vesicles derived from hiPSCs via the miR302b-3p/TGFβ/SMAD2 axis. BMC Med 2023; 21:412. [PMID: 37904135 PMCID: PMC10617123 DOI: 10.1186/s12916-023-03117-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/20/2023] [Accepted: 10/16/2023] [Indexed: 11/01/2023] Open
Abstract
BACKGROUND Cardiac fibrosis is one of the top killers among fibrotic diseases and continues to be a global unaddressed health problem. The lack of effective treatment combined with the considerable socioeconomic burden highlights the urgent need for innovative therapeutic options. Here, we evaluated the anti-fibrotic properties of extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSCs) that were cultured under various oxygen concentrations. METHODS EVs were isolated from three hiPSC lines cultured under normoxia (21% O2; EV-N) or reduced oxygen concentration (hypoxia): 3% O2 (EV-H3) or 5% O2 (EV-H5). The anti-fibrotic activity of EVs was tested in an in vitro model of cardiac fibrosis, followed by a detailed investigation of the underlying molecular mechanisms. Sequencing of EV miRNAs combined with bioinformatics analysis was conducted and a selected miRNA was validated using a miRNA mimic and inhibitor. Finally, EVs were tested in a mouse model of angiotensin II-induced cardiac fibrosis. RESULTS We provide evidence that an oxygen concentration of 5% enhances the anti-fibrotic effects of hiPS-EVs. These EVs were more effective in reducing pro-fibrotic markers in activated human cardiac fibroblasts, when compared to EV-N or EV-H3. We show that EV-H5 act through the canonical TGFβ/SMAD pathway, primarily via miR-302b-3p, which is the most abundant miRNA in EV-H5. Our results show that EV-H5 not only target transcripts of several profibrotic genes, including SMAD2 and TGFBR2, but also reduce the stiffness of activated fibroblasts. In a mouse model of heart fibrosis, EV-H5 outperformed EV-N in suppressing the inflammatory response in the host and by attenuating collagen deposition and reducing pro-fibrotic markers in cardiac tissue. CONCLUSIONS In this work, we provide evidence of superior anti-fibrotic properties of EV-H5 over EV-N or EV-H3. Our study uncovers that fine regulation of oxygen concentration in the cellular environment may enhance the anti-fibrotic effects of hiPS-EVs, which has great potential to be applied for heart regeneration.
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Affiliation(s)
- Milena Paw
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
| | - Agnieszka A Kusiak
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
- Doctoral School of Exact and Natural Sciences, Jagiellonian University, Kraków, Poland
| | - Kinga Nit
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
| | - Jacek J Litewka
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
- Doctoral School of Exact and Natural Sciences, Jagiellonian University, Kraków, Poland
| | - Marcin Piejko
- 3Rd Department of General Surgery, Jagiellonian University - Medical College, Kraków, Poland
| | - Dawid Wnuk
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
| | - Michał Sarna
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Biophysics, Jagiellonian University, Kraków, Poland
| | - Kinga Fic
- Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland
| | - Kinga B Stopa
- Doctoral School of Exact and Natural Sciences, Jagiellonian University, Kraków, Poland
- Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland
| | - Ruba Hammad
- Freiburg iPS Core Facility, Institute for Transfusion Medicine and Gene Therapy, Medical Center, University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), University of Freiburg, Freiburg, Germany
| | - Olga Barczyk-Woznicka
- Institute of Zoology and Biomedical Research, Department of Cell Biology and Imaging, Jagiellonian University, Kraków, Poland
| | - Toni Cathomen
- Freiburg iPS Core Facility, Institute for Transfusion Medicine and Gene Therapy, Medical Center, University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), University of Freiburg, Freiburg, Germany
| | - Ewa Zuba-Surma
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
| | - Zbigniew Madeja
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
| | - Paweł E Ferdek
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland
| | - Sylwia Bobis-Wozowicz
- Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University, Kraków, Poland.
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Levy D, Abadchi SN, Shababi N, Ravari MR, Pirolli NH, Bergeron C, Obiorah A, Mokhtari‐Esbuie F, Gheshlaghi S, Abraham JM, Smith IM, Powsner EH, Solomon TJ, Harmon JW, Jay SM. Induced Pluripotent Stem Cell-Derived Extracellular Vesicles Promote Wound Repair in a Diabetic Mouse Model via an Anti-Inflammatory Immunomodulatory Mechanism. Adv Healthc Mater 2023; 12:e2300879. [PMID: 37335811 PMCID: PMC10592465 DOI: 10.1002/adhm.202300879] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2023] [Revised: 06/07/2023] [Indexed: 06/21/2023]
Abstract
Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have recently been explored in clinical trials for treatment of diseases with complex pathophysiologies. However, production of MSC EVs is currently hampered by donor-specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self-renewing source for obtaining differentiated iPSC-derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Thus, it is initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, it is found that their vascularization bioactivity is similar and their anti-inflammatory bioactivity is superior to donor-matched iMSC EVs in cell-based assays. To supplement this initial in vitro bioactivity screen, a diabetic wound healing mouse model where both the pro-vascularization and anti-inflammatory activity of these EVs would be beneficial is employed. In this in vivo model, iPSC EVs more effectively mediate inflammation resolution within the wound bed. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated iPSCs as a source for therapeutic EV production with respect to both scalability and efficacy.
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Affiliation(s)
- Daniel Levy
- Fischell Department of BioengineeringUniversity of MarylandCollege ParkMD20742USA
| | | | - Niloufar Shababi
- Department of SurgeryJohns Hopkins University School of MedicineBaltimoreMD21224USA
| | | | - Nicholas H. Pirolli
- Fischell Department of BioengineeringUniversity of MarylandCollege ParkMD20742USA
| | - Cade Bergeron
- Fischell Department of BioengineeringUniversity of MarylandCollege ParkMD20742USA
| | - Angel Obiorah
- Fischell Department of BioengineeringUniversity of MarylandCollege ParkMD20742USA
| | | | - Shayan Gheshlaghi
- Department of SurgeryJohns Hopkins University School of MedicineBaltimoreMD21224USA
| | - John M. Abraham
- Department of SurgeryJohns Hopkins University School of MedicineBaltimoreMD21224USA
| | - Ian M. Smith
- Fischell Department of BioengineeringUniversity of MarylandCollege ParkMD20742USA
| | - Emily H. Powsner
- Fischell Department of BioengineeringUniversity of MarylandCollege ParkMD20742USA
| | - Talia J. Solomon
- Fischell Department of BioengineeringUniversity of MarylandCollege ParkMD20742USA
| | - John W. Harmon
- Department of SurgeryJohns Hopkins University School of MedicineBaltimoreMD21224USA
| | - Steven M. Jay
- Fischell Department of BioengineeringUniversity of MarylandCollege ParkMD20742USA
- Program in Molecular and Cell BiologyUniversity of MarylandCollege ParkMD20742USA
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Matos BMD, Stimamiglio MA, Correa A, Robert AW. Human pluripotent stem cell-derived extracellular vesicles: From now to the future. World J Stem Cells 2023; 15:453-465. [PMID: 37342215 PMCID: PMC10277970 DOI: 10.4252/wjsc.v15.i5.453] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/26/2022] [Revised: 02/14/2023] [Accepted: 04/13/2023] [Indexed: 05/26/2023] Open
Abstract
Extracellular vesicles (EVs) are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools. Accordingly, in various biological contexts, EVs are reported to engage in immune modulation, senescence, and cell proliferation and differentiation. Therefore, EVs could be key elements for potential off-the-shelf cell-free therapy. Little has been studied regarding EVs derived from human pluripotent stem cells (hPSC-EVs), even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability. In this review article, we provide an overview of studies using hPSC-EVs, focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs, how they are characterized, and applications already demonstrated. The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs’ prospective applications as PSC-derived cell-free therapy products.
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Affiliation(s)
- Bruno Moises de Matos
- Stem Cells Basic Biology Laboratory, Carlos Chagas Institute, Curitiba 81350010, Paraná, Brazil
| | | | - Alejandro Correa
- Stem Cells Basic Biology Laboratory, Carlos Chagas Institute, Curitiba 81350010, Paraná, Brazil
| | - Anny Waloski Robert
- Stem Cells Basic Biology Laboratory, Carlos Chagas Institute, Curitiba 81350010, Paraná, Brazil
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8
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Kronstadt SM, Van Heyningen LH, Aranda A, Jay SM. Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination. Cytotherapy 2023; 25:387-396. [PMID: 36599771 PMCID: PMC10006399 DOI: 10.1016/j.jcyt.2022.12.002] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2022] [Revised: 12/01/2022] [Accepted: 12/12/2022] [Indexed: 01/03/2023]
Abstract
Extracellular vesicles (EVs) are widely implicated as novel diagnostic and therapeutic modalities for a wide range of diseases. Thus, optimization of EV biomanufacturing is of high interest. In the course of developing parameters for a human embryonic kidney cells (HEK293T) EV production platform, we examined the combinatorial effects of cell culture conditions (i.e., static versus dynamic) and isolation techniques (i.e., ultracentrifugation versus tangential flow filtration versus size-exclusion chromatography) on functional characteristics of HEK293T EVs, including anti-inflammatory bioactivity using a well-established lipopolysaccharide-stimulated mouse macrophage model. We unexpectedly found that, depending on culture condition and isolation strategy, HEK293T EVs appeared to significantly suppress the secretion of pro-inflammatory cytokines (i.e., interleukin-6, RANTES [regulated upon activation, normal T cell expressed and secreted]) in the stimulated mouse macrophages. Further examination revealed that these results were most likely due to non-EV fetal bovine serum components in HEK293T EV preparations. Thus, future research assessing the anti-inflammatory effects of EVs should be designed to account for this phenomenon.
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Affiliation(s)
- Stephanie M Kronstadt
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, USA
| | | | - Amaya Aranda
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, USA
| | - Steven M Jay
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, USA; Program in Molecular and Cell Biology, University of Maryland, College Park, Maryland, USA.
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9
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Levy D, Abadchi SN, Shababi N, Ravari MR, Pirolli NH, Bergeron C, Obiorah A, Mokhtari-Esbuie F, Gheshlaghi S, Abraham JM, Smith IM, Powsner E, Solomon T, Harmon JW, Jay SM. Induced pluripotent stem cell-derived extracellular vesicles promote wound repair in a diabetic mouse model via an anti-inflammatory immunomodulatory mechanism. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.03.19.533334. [PMID: 36993554 PMCID: PMC10055496 DOI: 10.1101/2023.03.19.533334] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/31/2023]
Abstract
Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have recently been widely explored in clinical trials for treatment of diseases with complex pathophysiology. However, production of MSC EVs is currently hampered by donor-specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self-renewing source for obtaining differentiated iPSC-derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Thus, we initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, we found that their vascularization bioactivity was similar and their anti-inflammatory bioactivity was superior to donor-matched iMSC EVs in cell-based assays. To supplement this initial in vitro bioactivity screen, we employed a diabetic wound healing mouse model where both the pro-vascularization and anti-inflammatory activity of these EVs would be beneficial. In this in vivo model, iPSC EVs more effectively mediated inflammation resolution within the wound bed. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated iPSCs as a source for therapeutic EV production with respect to both scalability and efficacy.
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Affiliation(s)
- Daniel Levy
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA
| | | | - Niloufar Shababi
- Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA
| | - Mohsen Rouhani Ravari
- Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA
| | - Nicholas H. Pirolli
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA
| | - Cade Bergeron
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA
| | - Angel Obiorah
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA
| | - Farzad Mokhtari-Esbuie
- Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA
| | - Shayan Gheshlaghi
- Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA
| | - John M. Abraham
- Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA
| | - Ian M. Smith
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA
| | - Emily Powsner
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA
| | - Talia Solomon
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA
| | - John W. Harmon
- Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA
| | - Steven M. Jay
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA
- Program in Molecular and Cell Biology, University of Maryland, College Park, MD 20742, USA
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10
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Jin S, Lv Z, Kang L, Wang J, Tan C, Shen L, Wang L, Liu J. Next generation of neurological therapeutics: Native and bioengineered extracellular vesicles derived from stem cells. Asian J Pharm Sci 2022; 17:779-797. [PMID: 36600903 PMCID: PMC9800941 DOI: 10.1016/j.ajps.2022.10.002] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Revised: 09/20/2022] [Accepted: 10/10/2022] [Indexed: 11/19/2022] Open
Abstract
Extracellular vesicles (EVs)-based cell-free therapy, particularly stem cell-derived extracellular vesicles (SC-EVs), offers new insights into treating a series of neurological disorders and becomes a promising candidate for alternative stem cell regenerative therapy. Currently, SC-EVs are considered direct therapeutic agents by themselves and/or dynamic delivery systems as they have a similar regenerative capacity of stem cells to promote neurogenesis and can easily load many functional small molecules to recipient cells in the central nervous system. Meanwhile, as non-living entities, SC-EVs avoid the uncontrollability and manufacturability limitations of live stem cell products in vivo (e.g., low survival rate, immune response, and tumorigenicity) and in vitro (e.g., restricted sources, complex preparation processes, poor quality control, low storage, shipping instability, and ethical controversy) by strict quality control system. Moreover, SC-EVs can be engineered or designed to enhance further overall yield, increase bioactivity, improve targeting, and extend their half-life. Here, this review provides an overview on the biological properties of SC-EVs, and the current progress in the strategies of native or bioengineered SC-EVs for nerve injury repairing is presented. Then we further summarize the challenges of recent research and perspectives for successful clinical application to advance SC-EVs from bench to bedside in neurological diseases.
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Affiliation(s)
- Shilin Jin
- Stem Cell Clinical Research Center, National Joint Engineering Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
- Liaoning Key Laboratory of Frontier Technology of Stem Cell and Precision Medicine, Dalian Engineering Research Center for Genetic Variation Detection of Infectious Pathogenic Microorganisms, Dalian Innovation Institute of Stem Cell and Precision Medicine, Dalian 116085, China
| | - Zhongyue Lv
- Stem Cell Clinical Research Center, National Joint Engineering Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
- Liaoning Key Laboratory of Frontier Technology of Stem Cell and Precision Medicine, Dalian Engineering Research Center for Genetic Variation Detection of Infectious Pathogenic Microorganisms, Dalian Innovation Institute of Stem Cell and Precision Medicine, Dalian 116085, China
| | - Lin Kang
- Stem Cell Clinical Research Center, National Joint Engineering Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
- Liaoning Key Laboratory of Frontier Technology of Stem Cell and Precision Medicine, Dalian Engineering Research Center for Genetic Variation Detection of Infectious Pathogenic Microorganisms, Dalian Innovation Institute of Stem Cell and Precision Medicine, Dalian 116085, China
| | - Jiayi Wang
- Stem Cell Clinical Research Center, National Joint Engineering Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
- Liaoning Key Laboratory of Frontier Technology of Stem Cell and Precision Medicine, Dalian Engineering Research Center for Genetic Variation Detection of Infectious Pathogenic Microorganisms, Dalian Innovation Institute of Stem Cell and Precision Medicine, Dalian 116085, China
| | - Chengcheng Tan
- Stem Cell Clinical Research Center, National Joint Engineering Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
- Liaoning Key Laboratory of Frontier Technology of Stem Cell and Precision Medicine, Dalian Engineering Research Center for Genetic Variation Detection of Infectious Pathogenic Microorganisms, Dalian Innovation Institute of Stem Cell and Precision Medicine, Dalian 116085, China
| | - Liming Shen
- Stem Cell Clinical Research Center, National Joint Engineering Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
- Liaoning Key Laboratory of Frontier Technology of Stem Cell and Precision Medicine, Dalian Engineering Research Center for Genetic Variation Detection of Infectious Pathogenic Microorganisms, Dalian Innovation Institute of Stem Cell and Precision Medicine, Dalian 116085, China
| | - Liang Wang
- Stem Cell Clinical Research Center, National Joint Engineering Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
- Liaoning Key Laboratory of Frontier Technology of Stem Cell and Precision Medicine, Dalian Engineering Research Center for Genetic Variation Detection of Infectious Pathogenic Microorganisms, Dalian Innovation Institute of Stem Cell and Precision Medicine, Dalian 116085, China
| | - Jing Liu
- Stem Cell Clinical Research Center, National Joint Engineering Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
- Liaoning Key Laboratory of Frontier Technology of Stem Cell and Precision Medicine, Dalian Engineering Research Center for Genetic Variation Detection of Infectious Pathogenic Microorganisms, Dalian Innovation Institute of Stem Cell and Precision Medicine, Dalian 116085, China
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11
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Hallal S, Tűzesi Á, Grau GE, Buckland ME, Alexander KL. Understanding the extracellular vesicle surface for clinical molecular biology. J Extracell Vesicles 2022; 11:e12260. [PMID: 36239734 PMCID: PMC9563386 DOI: 10.1002/jev2.12260] [Citation(s) in RCA: 69] [Impact Index Per Article: 23.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2021] [Revised: 07/26/2022] [Accepted: 07/26/2022] [Indexed: 12/17/2022] Open
Abstract
Extracellular vesicles (EVs) are lipid-membrane enclosed nanoparticles that play significant roles in health and disease. EVs are abundant in body fluids and carry an array of molecules (proteins, lipids, nucleic acids and glycans) that reflect the identity and activity of their cell-of-origin. While the advent of high throughput omics technologies has allowed in-depth characterisation of EV compositions, how these molecular species are spatially distributed within EV structures is not well appreciated. This is particularly true of the EV surface where a plethora of molecules are reported to be both integral and peripherally associated to the EV membrane. This coronal layer or 'atmosphere' that surrounds the EV membrane contributes to a large, highly interactive and dynamic surface area that is responsible for facilitating EV interactions with the extracellular environment. The EV coronal layer harbours surface molecules that reflect the identity of parent cells, which is likely a highly valuable property in the context of diagnostic liquid biopsies. In this review, we describe the current understanding of the mechanical, electrostatic and molecular properties of the EV surface that offer significant biomarker potential and contribute to a highly dynamic interactome.
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Affiliation(s)
- Susannah Hallal
- Neurosurgery DepartmentChris O'Brien LifehouseCamperdownNSWAustralia,Brainstorm Brain Cancer Research, Brain and Mind CentreThe University of SydneyNSWAustralia,Neuropathology DepartmentRoyal Prince Alfred HospitalCamperdownNSWAustralia
| | - Ágota Tűzesi
- Brainstorm Brain Cancer Research, Brain and Mind CentreThe University of SydneyNSWAustralia,Neuropathology DepartmentRoyal Prince Alfred HospitalCamperdownNSWAustralia,School of Medical SciencesFaculty of Medicine & HealthThe University of SydneyCamperdownNSWAustralia
| | - Georges E. Grau
- School of Medical SciencesFaculty of Medicine & HealthThe University of SydneyCamperdownNSWAustralia
| | - Michael E. Buckland
- Brainstorm Brain Cancer Research, Brain and Mind CentreThe University of SydneyNSWAustralia,Neuropathology DepartmentRoyal Prince Alfred HospitalCamperdownNSWAustralia,School of Medical SciencesFaculty of Medicine & HealthThe University of SydneyCamperdownNSWAustralia
| | - Kimberley L. Alexander
- Neurosurgery DepartmentChris O'Brien LifehouseCamperdownNSWAustralia,Brainstorm Brain Cancer Research, Brain and Mind CentreThe University of SydneyNSWAustralia,Neuropathology DepartmentRoyal Prince Alfred HospitalCamperdownNSWAustralia,School of Medical SciencesFaculty of Medicine & HealthThe University of SydneyCamperdownNSWAustralia
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12
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Visan KS, Lobb RJ, Ham S, Lima LG, Palma C, Edna CPZ, Wu L, Gowda H, Datta KK, Hartel G, Salomon C, Möller A. Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles. J Extracell Vesicles 2022; 11:e12266. [PMID: 36124834 PMCID: PMC9486818 DOI: 10.1002/jev2.12266] [Citation(s) in RCA: 87] [Impact Index Per Article: 29.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2022] [Revised: 07/15/2022] [Accepted: 09/05/2022] [Indexed: 11/07/2022] Open
Abstract
Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.
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Affiliation(s)
- Kekoolani S. Visan
- Tumour Microenvironment LaboratoryQIMR Berghofer Medical Research InstituteHerstonQLDAustralia
| | - Richard J. Lobb
- Tumour Microenvironment LaboratoryQIMR Berghofer Medical Research InstituteHerstonQLDAustralia
- Centre for Personalized NanomedicineAustralian Institute for Bioengineering and Nanotechnology (AIBN)The University of QueenslandBrisbaneQLDAustralia
| | - Sunyoung Ham
- Tumour Microenvironment LaboratoryQIMR Berghofer Medical Research InstituteHerstonQLDAustralia
| | - Luize G. Lima
- Tumour Microenvironment LaboratoryQIMR Berghofer Medical Research InstituteHerstonQLDAustralia
| | - Carlos Palma
- Exosome Biology LaboratoryFaculty of Medicine and Biomedical SciencesCentre for Clinical DiagnosticsUniversity of Queensland Centre for Clinical ResearchRoyal Brisbane and Women's HospitalThe University of QueenslandBrisbaneQLDAustralia
| | - Chai Pei Zhi Edna
- Tumour Microenvironment LaboratoryQIMR Berghofer Medical Research InstituteHerstonQLDAustralia
| | - Li‐Ying Wu
- Tumour Microenvironment LaboratoryQIMR Berghofer Medical Research InstituteHerstonQLDAustralia
- School of Biomedical Sciences, Faculty of HealthQueensland University of TechnologyBrisbaneQLD4059Australia
| | - Harsha Gowda
- Cancer Precision Medicine LaboratoryQIMR Berghofer Medical Research InstituteHerstonQLDAustralia
| | - Keshava K. Datta
- Cancer Precision Medicine LaboratoryQIMR Berghofer Medical Research InstituteHerstonQLDAustralia
- Proteomics and Metabolomics PlatformLa Trobe UniversityBundooraVICAustralia
| | - Gunter Hartel
- Statistics UnitQIMR Berghofer Medical Research InstituteHerstonQLDAustralia
| | - Carlos Salomon
- Exosome Biology LaboratoryFaculty of Medicine and Biomedical SciencesCentre for Clinical DiagnosticsUniversity of Queensland Centre for Clinical ResearchRoyal Brisbane and Women's HospitalThe University of QueenslandBrisbaneQLDAustralia
- Departamento de InvestigaciónPostgrado y Educación Continua (DIPEC)Facultad de Ciencias de la SaludUniversidad del AlbaSantiagoChile
| | - Andreas Möller
- Tumour Microenvironment LaboratoryQIMR Berghofer Medical Research InstituteHerstonQLDAustralia
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13
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Carter N, Mathiesen AH, Miller N, Brown M, Colunga Biancatelli RML, Catravas JD, Dobrian AD. Endothelial cell-derived extracellular vesicles impair the angiogenic response of coronary artery endothelial cells. Front Cardiovasc Med 2022; 9:923081. [PMID: 35928931 PMCID: PMC9343725 DOI: 10.3389/fcvm.2022.923081] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2022] [Accepted: 06/28/2022] [Indexed: 12/17/2022] Open
Abstract
Cardiovascular disease (CVD) is the most prominent cause of death of adults in the United States with coronary artery disease being the most common type of CVD. Following a myocardial event, the coronary endothelium plays an important role in the recovery of the ischemic myocardium. Specifically, endothelial cells (EC) must be able to elicit a robust angiogenic response necessary for tissue revascularization and repair. However, local or distant cues may prevent effective revascularization. Extracellular vesicles (EV) are produced by all cells and endothelium is a rich source of EVs that have access to the main circulation thereby potentially impacting local and distant tissue function. Systemic inflammation associated with conditions such as obesity as well as the acute inflammatory response elicited by a cardiac event can significantly increase the EV release by endothelium and alter their miRNA, protein or lipid cargo. Our laboratory has previously shown that EVs released by adipose tissue endothelial cells exposed to chronic inflammation have angiostatic effects on naïve adipose tissue EC in vitro. Whether the observed effect is specific to EVs from adipose tissue endothelium or is a more general feature of the endothelial EVs exposed to pro-inflammatory cues is currently unclear. The objective of this study was to investigate the angiostatic effects of EVs produced by EC from the coronary artery and adipose microvasculature exposed to pro-inflammatory cytokines (PIC) on naïve coronary artery EC. We have found that EVs from both EC sources have angiostatic effects on the coronary endothelium. EVs produced by cells in a pro-inflammatory environment reduced proliferation and barrier function of EC without impacting cellular senescence. Some of these functional effects could be attributed to the miRNA cargo of EVs. Several miRNAs such as miR-451, let-7, or miR-23a impact on multiple pathways responsible for proliferation, cellular permeability and angiogenesis. Collectively, our data suggests that EVs may compete with pro-angiogenic cues in the ischemic myocardium therefore slowing down the repair response. Acute treatments with inhibitors that prevent endogenous EV release immediately after an ischemic event may contribute to better efficacy of therapeutic approaches using functionalized exogenous EVs or other pro-angiogenic approaches.
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Affiliation(s)
- Nigeste Carter
- Department of Physiological Science, Eastern Virginia Medical School, Norfolk, VA, United States
| | - Allison H. Mathiesen
- Department of Physiological Science, Eastern Virginia Medical School, Norfolk, VA, United States
| | - Noel Miller
- Department of Physiological Science, Eastern Virginia Medical School, Norfolk, VA, United States
| | - Michael Brown
- Department of Physiological Science, Eastern Virginia Medical School, Norfolk, VA, United States
| | | | - John D. Catravas
- Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, United States
- School of Medical Diagnostic and Translational Sciences, College of Health Sciences, Old Dominion University, Norfolk, VA, United States
| | - Anca D. Dobrian
- Department of Physiological Science, Eastern Virginia Medical School, Norfolk, VA, United States
- *Correspondence: Anca D. Dobrian,
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14
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Quaglia M, Merlotti G, Colombatto A, Bruno S, Stasi A, Franzin R, Castellano G, Grossini E, Fanelli V, Cantaluppi V. Stem Cell-Derived Extracellular Vesicles as Potential Therapeutic Approach for Acute Kidney Injury. Front Immunol 2022; 13:849891. [PMID: 35359949 PMCID: PMC8960117 DOI: 10.3389/fimmu.2022.849891] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2022] [Accepted: 02/15/2022] [Indexed: 12/12/2022] Open
Abstract
Acute kidney injury is a frequent complication of hospitalized patients and significantly increases morbidity and mortality, worsening costs and length of hospital stay. Despite this impact on healthcare system, treatment still remains only supportive (dialysis). Stem cell-derived extracellular vesicles are a promising option as they recapitulate stem cells properties, overcoming safety issues related to risks or rejection or aberrant differentiation. A growing body of evidence based on pre-clinical studies suggests that extracellular vesicles may be effective to treat acute kidney injury and to limit fibrosis through direct interference with pathogenic mechanisms of vascular and tubular epithelial cell damage. We herein analyze the state-of-the-art knowledge of therapeutic approaches with stem cell-derived extracellular vesicles for different forms of acute kidney injury (toxic, ischemic or septic) dissecting their cytoprotective, regenerative and immunomodulatory properties. We also analyze the potential impact of extracellular vesicles on the mechanisms of transition from acute kidney injury to chronic kidney disease, with a focus on the pivotal role of the inhibition of complement cascade in this setting. Despite some technical limits, nowadays the development of therapies based on stem cell-derived extracellular vesicles holds promise as a new frontier to limit acute kidney injury onset and progression.
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Affiliation(s)
- Marco Quaglia
- Nephrology and Kidney Transplantation Unit, "Maggiore della Carità" University Hospital, Department of Translational Medicine, Translational Research on Autoimmune and Allergic Disease (CAAD), University of Piemonte Orientale (UPO), Novara, Italy
| | - Guido Merlotti
- Nephrology and Kidney Transplantation Unit, "Maggiore della Carità" University Hospital, Department of Translational Medicine, Translational Research on Autoimmune and Allergic Disease (CAAD), University of Piemonte Orientale (UPO), Novara, Italy
| | - Andrea Colombatto
- Nephrology and Kidney Transplantation Unit, "Maggiore della Carità" University Hospital, Department of Translational Medicine, Translational Research on Autoimmune and Allergic Disease (CAAD), University of Piemonte Orientale (UPO), Novara, Italy
| | - Stefania Bruno
- Department of Medical Sciences, University of Torino, Torino, Italy
| | - Alessandra Stasi
- Nephrology, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Rossana Franzin
- Nephrology, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Giuseppe Castellano
- Nephrology, Dialysis and Kidney Transplantation Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy
| | - Elena Grossini
- Laboratory of Physiology, Department of Translational Medicine, Translational Research on Autoimmune and Allergic Disease (CAAD), University of Piemonte Orientale, Novara, Italy
| | - Vito Fanelli
- Department of Anesthesiology and Intensive Care, University of Torino, Torino, Italy
| | - Vincenzo Cantaluppi
- Nephrology and Kidney Transplantation Unit, "Maggiore della Carità" University Hospital, Department of Translational Medicine, Translational Research on Autoimmune and Allergic Disease (CAAD), University of Piemonte Orientale (UPO), Novara, Italy
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15
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Gomes FG, Andrade AC, Wolf M, Hochmann S, Krisch L, Maeding N, Regl C, Poupardin R, Ebner-Peking P, Huber CG, Meisner-Kober N, Schallmoser K, Strunk D. Synergy of Human Platelet-Derived Extracellular Vesicles with Secretome Proteins Promotes Regenerative Functions. Biomedicines 2022; 10:238. [PMID: 35203448 PMCID: PMC8869293 DOI: 10.3390/biomedicines10020238] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2021] [Revised: 01/17/2022] [Accepted: 01/19/2022] [Indexed: 11/29/2022] Open
Abstract
Platelet-rich plasma is a promising regenerative therapeutic with controversial efficacy. We and others have previously demonstrated regenerative functions of human platelet lysate (HPL) as an alternative platelet-derived product. Here we separated extracellular vesicles (EVs) from soluble factors of HPL to understand the mode of action during skin-organoid formation and immune modulation as model systems for tissue regeneration. HPL-EVs were isolated by tangential-flow filtration (TFF) and further purified by size-exclusion chromatography (SEC) separating EVs from (lipo)protein-enriched soluble fractions. We characterized samples by tunable resistive pulse sensing, western blot, tandem mass-tag proteomics and super-resolution microscopy. We evaluated EV function during angiogenesis, wound healing, organoid formation and immune modulation. We characterized EV enrichment by TFF and SEC according to MISEV2018 guidelines. Proteomics showed three major clusters of protein composition separating TSEC-EVs from HPL clustering with TFF soluble fractions and TFF-EVs clustering with TSEC soluble fractions, respectively. HPL-derived TFF-EVs promoted skin-organoid formation and inhibited T-cell proliferation more efficiently than TSEC-EVs or TSEC-soluble fractions. Recombining TSEC-EVs with TSEC soluble fractions re-capitulated TFF-EV effects. Zeta potential and super-resolution imaging further evidenced protein corona formation on TFF-EVs. Corona depletion on SEC-EVs could be artificially reconstituted by TSEC late fraction add-back. In contrast to synthetic nanoparticles, which commonly experience reduced function after corona formation, the corona-bearing EVs displayed improved functionality. We conclude that permissive isolation technology, such as TFF, and better understanding of the mechanism of EV corona function are required to realize the complete potential of platelet-based regenerative therapies.
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Affiliation(s)
- Fausto Gueths Gomes
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria;
| | - André Cronemberger Andrade
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Martin Wolf
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Sarah Hochmann
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Linda Krisch
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria;
| | - Nicole Maeding
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Christof Regl
- Department for Biosciences and Medical Biology, Paris Lodron University, 5020 Salzburg, Austria; (C.R.); (C.G.H.); (N.M.-K.)
| | - Rodolphe Poupardin
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Patricia Ebner-Peking
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Christian G. Huber
- Department for Biosciences and Medical Biology, Paris Lodron University, 5020 Salzburg, Austria; (C.R.); (C.G.H.); (N.M.-K.)
| | - Nicole Meisner-Kober
- Department for Biosciences and Medical Biology, Paris Lodron University, 5020 Salzburg, Austria; (C.R.); (C.G.H.); (N.M.-K.)
| | - Katharina Schallmoser
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria;
| | - Dirk Strunk
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
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16
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Binder HM, Maeding N, Wolf M, Cronemberger Andrade A, Vari B, Krisch L, Gomes FG, Blöchl C, Muigg K, Poupardin R, Raninger AM, Heuser T, Obermayer A, Ebner-Peking P, Pleyer L, Greil R, Huber CG, Schallmoser K, Strunk D. Scalable Enrichment of Immunomodulatory Human Acute Myeloid Leukemia Cell Line-Derived Extracellular Vesicles. Cells 2021; 10:3321. [PMID: 34943829 PMCID: PMC8699161 DOI: 10.3390/cells10123321] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Revised: 11/17/2021] [Accepted: 11/24/2021] [Indexed: 12/15/2022] Open
Abstract
Acute myeloid leukemia (AML) cells can secrete trophic factors, including extracellular vesicles (EVs), instructing the stromal leukemic niche. Here, we introduce a scalable workflow for purification of immunomodulatory AML-EVs to compare their phenotype and function to the parental AML cells and their secreted soluble factors. AML cell lines HL-60, KG-1, OCI-AML3, and MOLM-14 released EVs with a peak diameter of approximately 80 nm in serum-free particle-reduced medium. We enriched EVs >100x using tangential flow filtration (TFF) and separated AML-derived soluble factors and cells in parallel. EVs were characterized by electron microscopy, immunoblotting, and flow cytometry, confirming the double-membrane morphology, purity and identity. AML-EVs showed significant enrichment of immune response and leukemia-related pathways in tandem mass-tag proteomics and a significant dose-dependent inhibition of T cell proliferation, which was not observed with AML cells or their soluble factors. Furthermore, AML-EVs dose-dependently reduced NK cell lysis of third-party K-562 leukemia targets. This emphasizes the peculiar role of AML-EVs in leukemia immune escape and indicates novel EV-based targets for therapeutic interventions.
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Affiliation(s)
- Heide-Marie Binder
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
| | - Nicole Maeding
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
| | - Martin Wolf
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
| | - André Cronemberger Andrade
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
| | - Balazs Vari
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
| | - Linda Krisch
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria;
| | - Fausto Gueths Gomes
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
| | - Constantin Blöchl
- Department of Biosciences, Paris Lodron University, 5020 Salzburg, Austria; (C.B.); (A.O.); (C.G.H.)
| | - Katharina Muigg
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
| | - Rodolphe Poupardin
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
| | - Anna M. Raninger
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
| | - Thomas Heuser
- Vienna BioCenter Core Facilities GmbH, 1030 Vienna, Austria;
| | - Astrid Obermayer
- Department of Biosciences, Paris Lodron University, 5020 Salzburg, Austria; (C.B.); (A.O.); (C.G.H.)
| | - Patricia Ebner-Peking
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
| | - Lisa Pleyer
- 3rd Medical Department with Hematology, Medical Oncology, Rheumatology and Infectiology, Paracelsus Medical University, 5020 Salzburg, Austria; (L.P.); (R.G.)
- Salzburg Cancer Research Institute (SCRI) Center for Clinical Cancer and Immunology Trials (CCCIT) and Cancer Cluster Salzburg (CCS), 5020 Salzburg, Austria
- Austrian Group for Medical Tumor Therapy (AGMT) Study Group, 1180 Vienna, Austria
| | - Richard Greil
- 3rd Medical Department with Hematology, Medical Oncology, Rheumatology and Infectiology, Paracelsus Medical University, 5020 Salzburg, Austria; (L.P.); (R.G.)
- Salzburg Cancer Research Institute (SCRI) Center for Clinical Cancer and Immunology Trials (CCCIT) and Cancer Cluster Salzburg (CCS), 5020 Salzburg, Austria
- Austrian Group for Medical Tumor Therapy (AGMT) Study Group, 1180 Vienna, Austria
| | - Christian G. Huber
- Department of Biosciences, Paris Lodron University, 5020 Salzburg, Austria; (C.B.); (A.O.); (C.G.H.)
| | - Katharina Schallmoser
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria;
| | - Dirk Strunk
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (H.-M.B.); (N.M.); (M.W.); (A.C.A.); (B.V.); (L.K.); (F.G.G.); (K.M.); (R.P.); (A.M.R.); (P.E.-P.)
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17
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Poupardin R, Wolf M, Strunk D. Adherence to minimal experimental requirements for defining extracellular vesicles and their functions. Adv Drug Deliv Rev 2021; 176:113872. [PMID: 34284058 DOI: 10.1016/j.addr.2021.113872] [Citation(s) in RCA: 51] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Revised: 07/09/2021] [Accepted: 07/12/2021] [Indexed: 02/07/2023]
Abstract
Rigorous measures are required to cope with the advance of extracellular vesicle (EV) research, from 183 studies published in 2012 to 2,309 studies published in 2020. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines in 2014, updated in 2018, for assuring and improving EV research quality. We performed a systematic review using a text mining approach to assess adherence to MISEV criteria. A keyword search was conducted in 5,093 accessible publications over the period 2012-2020 and analyzed the methodology used for EV isolation and characterization. We found a significant improvement over the years particularly regarding EV characterization where recent papers used a higher number of methods and EV markers to check for quantity and purity. Interestingly, we also found that EV papers using more methods and EV markers were cited more frequently. Papers citing MISEV criteria were more prone to use a higher number of characterization methods. We therefore established a concise checklist summarizing MISEV criteria to support EV researchers towards reaching the highest standards in the field.
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Affiliation(s)
- Rodolphe Poupardin
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI - TReCS), Paracelsus Medical University (PMU), Salzburg, Austria
| | - Martin Wolf
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI - TReCS), Paracelsus Medical University (PMU), Salzburg, Austria
| | - Dirk Strunk
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI - TReCS), Paracelsus Medical University (PMU), Salzburg, Austria.
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18
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Krisch L, Brachtl G, Hochmann S, Andrade AC, Oeller M, Ebner-Peking P, Schallmoser K, Strunk D. Improving Human Induced Pluripotent Stem Cell-Derived Megakaryocyte Differentiation and Platelet Production. Int J Mol Sci 2021; 22:8224. [PMID: 34360992 PMCID: PMC8348107 DOI: 10.3390/ijms22158224] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Revised: 07/22/2021] [Accepted: 07/28/2021] [Indexed: 11/17/2022] Open
Abstract
Several protocols exist for generating megakaryocytes (MKs) and platelets from human induced pluripotent stem cells (hiPSCs) with limited efficiency. We observed previously that mesoderm induction improved endothelial and stromal differentiation. We, therefore, hypothesized that a protocol modification prior to hemogenic endothelial cell (HEC) differentiation will improve MK progenitor (MKP) production and increase platelet output. We further asked if basic media composition affects MK maturation. In an iterative process, we first compared two HEC induction protocols. We found significantly more HECs using the modified protocol including activin A and CHIR99021, resulting in significantly increased MKs. MKs released comparable platelet amounts irrespective of media conditions. In a final validation phase, we obtained five-fold more platelets per hiPSC with the modified protocol (235 ± 84) compared to standard conditions (51 ± 15; p < 0.0001). The regenerative potency of hiPSC-derived platelets was compared to adult donor-derived platelets by profiling angiogenesis-related protein expression. Nineteen of 24 angiogenesis-related proteins were expressed equally, lower or higher in hiPSC-derived compared to adult platelets. The hiPSC-platelet's coagulation hyporeactivity compared to adult platelets was confirmed by thromboelastometry. Further stepwise improvement of hiPSC-platelet production will, thus, permit better identification of platelet-mediated regenerative mechanisms and facilitate manufacture of sufficient amounts of functional platelets for clinical application.
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Affiliation(s)
- Linda Krisch
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (L.K.); (G.B.); (S.H.); (A.C.A.); (P.E.-P.)
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (M.O.); (K.S.)
| | - Gabriele Brachtl
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (L.K.); (G.B.); (S.H.); (A.C.A.); (P.E.-P.)
| | - Sarah Hochmann
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (L.K.); (G.B.); (S.H.); (A.C.A.); (P.E.-P.)
| | - André Cronemberger Andrade
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (L.K.); (G.B.); (S.H.); (A.C.A.); (P.E.-P.)
| | - Michaela Oeller
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (M.O.); (K.S.)
| | - Patricia Ebner-Peking
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (L.K.); (G.B.); (S.H.); (A.C.A.); (P.E.-P.)
| | - Katharina Schallmoser
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (M.O.); (K.S.)
| | - Dirk Strunk
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (L.K.); (G.B.); (S.H.); (A.C.A.); (P.E.-P.)
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