Name: protocol for microrna transfer into adult bone marrow-derived hematopoietic stem cells to enable cell engineering combined with magnetic targeting
Uploaded: 2018-06-18T13:00:00
Description: Watch this Scientific Journal Video about Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting at JoVE.com

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).

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.
<ol>
	<li>CD133+ SC isolation
	<ol...

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,...

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.
Despite their high potential demonstrated in more than 30 approved clinical trials (ClinicalTrails.gov), their therapeutic outcome is still under controversial discussion4. Indeed, a clinical application of SCs is hampered by low retention in the organ of interest and massive initial cell death5,8,9. Additional engineering of CD133+ SCs prior transplantation could help overcome these challenges.
One prerequisite for an efficient cell therapy would be the reduction of the massive initial cell death to enhance the engraftment of therapeutic relevant cells10. Current studies demonstrated an immense cell loss of 90&#8211;99% in highly perfused organs such as the brain and heart during the first 1&#8211;2 h, independent of the transplanted cell type or application route11,12,13,14,15,16,17,18,19,20,21. SC labeling using magnetic nanoparticles (MNPs) enables an innovative non-invasive strategy to target cells to the site of interest22,23,24,25,26 and simultaneously allows cell monitoring using MRI27 and magnetic particle imaging (MPI). The most efficient in vivo studies applying magnetized cell targeting used cell retention after local administration in preference to cell guidance after intravenous injection23,24,28. Therefore, our group designed a delivery system consisting of superparamagnetic iron oxide nanoparticles29. With this technique, CD133+ SCs and human umbilical vein endothelial cells (HUVECs) could efficiently be targeted, as demonstrated by in vitro attempts30,31.
Another hurdle for SC therapies is the hostile inflammatory environment of the affected tissue after transplantation, which contributes to the initial cell death32. In addition to several pre-conditioning studies, the application of therapeutic relevant miRs was tested33; it has been successfully demonstrated that anti-apoptotic miRs inhibit apoptosis in vitro and enhance cell engraftment in vivo33. These small molecules, composed of 20&#8211;25 nucleotides, play a crucial role as posttranscriptional modulators of messenger RNAs (mRNAs), and thus affect stem cell fate and behavior34. Moreover, the exogenous introduction of miRs avoid the undesired stable integration into the host genome34.
Current attempts for efficient introduction of nucleic acids (NAs) into primary SCs are mostly based on recombinant viruses8,35. Despite the high transfection efficiency, recombinant virus manipulation presents a major obstacle for a bench-to-bedside translation, e.g., uncontrollable gene expression, pathogenicity, immunogenicity, and insertional mutagenesis35,36. Therefore, non-viral delivery systems such as polymer-based constructs are critical to develop. Among those, polyethylenimine (PEI) represents a valid delivery vehicle offering benefits for miRs such as NA condensation to protect from degradation, cellular uptake, and intracellular release through endosomal escape37,38. Furthermore, miR-PEI complexes demonstrated a high biocompatibility in clinical trials39. Therefore, our delivery system consists of a biotinylated branched 25 kDa PEI bound to a streptavidin-coated MNP-core30,31,40.
In this manuscript, we present a comprehensive protocol describing (i) the manual isolation of CD133+ SC from human bone marrow (BM) donation with a detailed characterization of the SC product and (ii) an efficient and gentle transfection strategy of a magnetically non-viral polymer-based delivery system for genetic engineering of CD133+ SCs using miRs. CD133+ SCs are isolated and magnetically enriched from human sternal BM aspirates using a surface antibody-based magnetic-activated cell sorting (MACS) system. Afterwards, the cell viability as well as the cell purity are analyzed using flow cytometry. Subsequently, miR/PEI/MNP complexes are prepared and CD133+ SCs are transfected. 18 h after transfection, the uptake efficiency and the impact of transfection on SC marker expression and cell viability are analyzed. Moreover, evaluation of the intracellular distribution of the transfection complex compounds is performed using four-color labeling and structured illumination microscopy (SIM).

<table>
	<tbody>
		<tr>
			<td>7-AAD</td>
			<td>BD Biosciences</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid with Methylene Blue</td>
			<td>Stemcell Technologies</td>
			<td>7060</td>
			<td>3%</td>
		</tr>
		<tr>
			<td>anti-CD133/2-PE (clone: 293C3)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-090-853</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD34-FITC (clone: AC136)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-081-001</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45-APC-H7 (clone: 2D1)</td>
			<td>BD Biosciences</td>
			<td>560178</td>
			<td></td>
		</tr>
		<tr>
			<td>rhodamine dye; Atto 565 dye conjugated to biotin</td>
			<td>ATTO-TEC GmbH</td>
			<td>AD 565-71</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS LSRII flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva Software 6.1.2</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich GmbH</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>CD133 antibody-linked superparamagnetic iron dextran particles; CD133 MicroBead Kit</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-049</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase B</td>
			<td>Roche Diagnostics GmbH</td>
			<td>11088831001</td>
			<td></td>
		</tr>
		<tr>
			<td>counting chamber</td>
			<td>Paul Marienfeld GmbH &#38; Co. KG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanine 3 dye labelled precursor miR; Cy3 Dye-Labeled Pre-miR Negative Control #1</td>
			<td>Ambion</td>
			<td>AM17120</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanine 5 dye miR labelling kit; Cy5 dye Label IT miRNA Labeling Kit</td>
			<td>Mirus Bio</td>
			<td>MIR 9650</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Roche Diagnostics GmbH</td>
			<td>10104159001</td>
			<td>(100 U/mL)</td>
		</tr>
		<tr>
			<td>ELYRA PS.1 LSM 780 confocal microscope</td>
			<td>Carl Zeiss Jena GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FcR Blocking Reagent, human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-059-901</td>
			<td></td>
		</tr>
		<tr>
			<td>bright green protein labeling kit; Oregon Green 488 Protein Labeling Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>O10241</td>
			<td></td>
		</tr>
		<tr>
			<td>aqueous mounting medium; Fluoroshield</td>
			<td>Sigma-Aldrich GmbH</td>
			<td>F6182</td>
			<td></td>
		</tr>
		<tr>
			<td>density gradient centrifugation tube; Leukosep Centrifuge Tube</td>
			<td>Greiner Bio-One</td>
			<td>89048-932</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS magnet holder; MACS MultiStand</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS pre-separation filter</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-041-407</td>
			<td>30 &#181;m</td>
		</tr>
		<tr>
			<td>MACS separation column (MS / LS)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-042-201 / 130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS permanent magnet; MACS Separator</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HV PVDF Filter</td>
			<td>Merck</td>
			<td>SLHV013SL</td>
			<td>0.45 &#956;m</td>
		</tr>
		<tr>
			<td>mouse IgG 2b-PE</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-092-215</td>
			<td></td>
		</tr>
		<tr>
			<td>amine reactive dye; Near-IR LIVE/DEAD Fixable Dead Cell Stain Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>L10119</td>
			<td></td>
		</tr>
		<tr>
			<td>human lymphocyte separating medium; Pancoll</td>
			<td>Pan Biotech GmbH</td>
			<td>P04-60500</td>
			<td>density: 1.077 g/mL</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Pan Biotech GmbH</td>
			<td>P04-53500</td>
			<td>without Ca and Mg</td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Sigma-Aldrich GmbH</td>
			<td>408727</td>
			<td>branched; 25 kDa</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>100 U/mL, 100 &#956;g/mL</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Merck Schuchardt OHG</td>
			<td>1040051000</td>
			<td></td>
		</tr>
		<tr>
			<td>unlabelled precursor miR; Pre-miR miRNA Precursor Negative Control #1</td>
			<td>Ambion</td>
			<td>AM17110</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>eBioscience</td>
			<td>00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse decontamination solution; RNaseZap</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>human lymphocyte medium; Roswell Park Memorial Institute (RPMI) 1640 medium</td>
			<td>Pan Biotech GmbH</td>
			<td>P04-16500</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human cytokine supplement; StemSpan CC100</td>
			<td>Stemcell Technologies</td>
			<td>2690</td>
			<td></td>
		</tr>
		<tr>
			<td>serum-free haematopoietic cell expansion medium; StemSpan H3000</td>
			<td>Stemcell Technologies</td>
			<td>9800</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin MagneSphere Paramagnetic Particles</td>
			<td>Promega Corporation</td>
			<td>Z5481</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Sigma-Aldrich GmbH</td>
			<td>T8154</td>
			<td>0.4 %</td>
		</tr>
		<tr>
			<td>UltraPure EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575020</td>
			<td>0.5 M; pH 8.0</td>
		</tr>
		<tr>
			<td>ZEN2011 software</td>
			<td>Carl Zeiss Jena GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td colspan="2">Thermo Fisher Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonorex RK 100 SH sonicating water bath</td>
			<td>Bandelin electronic</td>
			<td></td>
			<td>Ultrasonic nominal output: 80 W; Ultrasonic frequency: 35 kHz</td>
		</tr>
	</tbody>
</table>

protocol for microrna transfer into adult bone marrow-derived hematopoietic stem cells to enable cell engineering combined with magnetic targeting

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&#8211;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).

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...

Watch this Scientific Journal Video about Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting at JoVE.com

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

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.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

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