This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.
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Method Article
Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting
* These authors contributed equally
In This Article
Summary
Abstract
While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80–90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).
Introduction
CD133+ SCs represent a heterogeneous stem and progenitor cell population with promising potential for regenerative medicine. Their hematopoietic, endothelial, and myogenic differentiation potential1,2,3 enables the CD133+ cells, e.g., to contribute to neovascularization processes through differentiation into newly forming vessels and activation of pro-angiogenic signaling by paracrine mechanisms4,5,6,7.
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Protocol
Sternal human BM for cell isolation was obtained from informed donors, who gave their written consent to use their samples for research according to the Declaration of Helsinki. The ethical committee of the University of Rostock has approved the presented study (reg. No. A 2010 23, prolonged in 2015).
1. Cell Preparation
NOTE: Use heparin sodium (250 IU/mL BM) to prevent coagulation for BM examination.
- CD133+ SC isolation
- Preparation of required solutions
- Prepare phosphate buffered saline (PBS)/ ethylenediaminetetraacetic acid (EDTA) stock solution....
- Preparation of required solutions
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Results
The presented protocol describes a manual isolation and magnetic enrichment of human BM-derived CD133+ SCs with a subsequent virus independent cell engineering strategy, as a non-invasive technology for in vitro cell manipulation and in vivo monitoring tool.
This three-step isolation technology permits a separation of MNCs from the pre-digested sternal BM through density gradient centrifugation. Afte.......
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Discussion
In recent years, CD133+ SCs have emerged as a promising cell population for SC-based therapies as evidenced by several phase I, II, and III clinical trials43,44,45,46,47,48,49,50,51,.......
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Disclosures
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the Federal Ministry of Education and Research Germany (FKZ 0312138A and FKZ 316159), the State Mecklenburg-Western Pomerania with EU Structural Funds (ESF/IVWM-B34-0030/10 and ESF/IVBM-B35-0010/12), and the DFG (DA1296/2-1), the German Heart Foundation (F/01/12), the BMBF (VIP+ 00240) and the DAMP Foundation. In addition, F.H. and P.M. are supported by the FORUN Program of Rostock University Medical Centre (889001).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 7-AAD | BD Biosciences | 559925 | |
| Acetic Acid with Methylene Blue | Stemcell Technologies | 7060 | 3% |
| anti-CD133/2-PE (clone: 293C3) | Miltenyi Biotec GmbH | 130-090-853 | |
| anti-CD34-FITC (clone: AC136) | Miltenyi Biotec GmbH | 130-081-001 | |
| anti-CD45-APC-H7 (clone: 2D1) | BD Biosciences | 560178 | |
| rhodamine dye; Atto 565 dye conjugated to biotin | ATTO-TEC GmbH | AD 565-71 | |
| BD FACS LSRII flow cytometer | BD Biosciences | ||
| BD FACSDiva Software 6.1.2 | BD Biosciences | ||
| BSA | Sigma-Aldrich GmbH | A7906 | |
| CD133 antibody-linked superparamagnetic iron dextran particles; CD133 MicroBead Kit | Miltenyi Biotec GmbH | 130-097-049 | |
| collagenase B | Roche Diagnostics GmbH | 11088831001 | |
| counting chamber | Paul Marienfeld GmbH & Co. KG | ||
| Cyanine 3 dye labelled precursor miR; Cy3 Dye-Labeled Pre-miR Negative Control #1 | Ambion | AM17120 | |
| Cyanine 5 dye miR labelling kit; Cy5 dye Label IT miRNA Labeling Kit | Mirus Bio | MIR 9650 | |
| DNAse I | Roche Diagnostics GmbH | 10104159001 | (100 U/mL) |
| ELYRA PS.1 LSM 780 confocal microscope | Carl Zeiss Jena GmbH | ||
| FcR Blocking Reagent, human | Miltenyi Biotec GmbH | 130-059-901 | |
| bright green protein labeling kit; Oregon Green 488 Protein Labeling Kit | Thermo Fisher Scientific | O10241 | |
| aqueous mounting medium; Fluoroshield | Sigma-Aldrich GmbH | F6182 | |
| density gradient centrifugation tube; Leukosep Centrifuge Tube | Greiner Bio-One | 89048-932 | |
| MACS magnet holder; MACS MultiStand | Miltenyi Biotec GmbH | 130-042-303 | |
| MACS pre-separation filter | Miltenyi Biotec GmbH | 130-041-407 | 30 µm |
| MACS separation column (MS / LS) | Miltenyi Biotec GmbH | 130-042-201 / 130-042-401 | |
| MACS permanent magnet; MACS Separator | Miltenyi Biotec GmbH | 130-042-302 | |
| Millex-HV PVDF Filter | Merck | SLHV013SL | 0.45 μm |
| mouse IgG 2b-PE | Miltenyi Biotec GmbH | 130-092-215 | |
| amine reactive dye; Near-IR LIVE/DEAD Fixable Dead Cell Stain Kit | Thermo Fisher Scientific | L10119 | |
| human lymphocyte separating medium; Pancoll | Pan Biotech GmbH | P04-60500 | density: 1.077 g/mL |
| PBS | Pan Biotech GmbH | P04-53500 | without Ca and Mg |
| PEI | Sigma-Aldrich GmbH | 408727 | branched; 25 kDa |
| Penicillin/Streptomycin | Thermo Fisher Scientific | 15140122 | 100 U/mL, 100 μg/mL |
| PFA | Merck Schuchardt OHG | 1040051000 | |
| unlabelled precursor miR; Pre-miR miRNA Precursor Negative Control #1 | Ambion | AM17110 | |
| RBC lysis buffer | eBioscience | 00-4333-57 | |
| RNAse decontamination solution; RNaseZap | Thermo Fisher Scientific | AM9780 | |
| human lymphocyte medium; Roswell Park Memorial Institute (RPMI) 1640 medium | Pan Biotech GmbH | P04-16500 | |
| recombinant human cytokine supplement; StemSpan CC100 | Stemcell Technologies | 2690 | |
| serum-free haematopoietic cell expansion medium; StemSpan H3000 | Stemcell Technologies | 9800 | |
| Streptavidin MagneSphere Paramagnetic Particles | Promega Corporation | Z5481 | |
| Trypan Blue solution | Sigma-Aldrich GmbH | T8154 | 0.4 % |
| UltraPure EDTA | Thermo Fisher Scientific | 15575020 | 0.5 M; pH 8.0 |
| ZEN2011 software | Carl Zeiss Jena GmbH | ||
| NanoDrop 1000 Spectrophotometer | Thermo Fisher Scientific | ||
| Sonorex RK 100 SH sonicating water bath | Bandelin electronic | Ultrasonic nominal output: 80 W; Ultrasonic frequency: 35 kHz |
References
- Meregalli, M., Farini, A., Belicchi, M., Torrente, Y. CD133(+) cells isolated from various sources and their role in future clinical perspectives. Expert opinion on biological therapy. 10 (11), 1521-1528 (2010).
- Lee, S., Yoon, Y. -S.
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