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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

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DOI:

10.3791/57474

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June 18th, 2018

 ,  ,  ,  , 
1Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), Department of Cardiac Surgery, Rostock University Medical Center, 2Department Life, Light and Matter of the Interdisciplinary Faculty, Rostock University

* These authors contributed equally

In This Article

Summary

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.

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.

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 90–99% in highly perfused organs such as the brain and heart during the first 1–2 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 20–25 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).

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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.

  1. CD133+ SC isolation
    1. Preparation of required solutions
      1. Prepare phosphate buffered saline (PBS)/ ethylenediaminetetraacetic acid (EDTA) stock solution (2 mM) by mixing 996 mL of 1x PBS with 4 mL of EDTA (0.5 M). Vigorously mix the solution and filtrate using a 0.45 µm filter. Store at room temperature (RT).
      2. Prepare the MACS buffer by mixing 995 mL of 2 mM PBS/EDTA with 5 g of bovine serum albumin (BSA). Vigorously mix the solution and filtrate using a 0.45 µm filter. Store at 4 °C.
      3. Prepare collagenase B stock solution (40 mg/mL) by diluting 500 mg of collagenase B (from Clostridium histolyticum) in 12.5 mL of PBS. Vigorously mix the solution, make aliquots of 350 µL, and store at -20 °C.
      4. Prepare deoxyribonuclease (DNAse) I stock solution (10 mg/mL) by diluting 100 mg of DNase I (from bovine pancreas) in 10 mL of PBS. Vigorously mix the solution, make aliquots of 350 µL, and store at -20 °C.
    2. Enzymatic digestion of human BM
      1. Pre-warm the human lymphocyte medium to RT and thaw enzymes (1 aliquot each of collagenase B and DNAse per 20 mL BM).
      2. Collect the BM from syringes into a 50 mL conical tube and mix gently. Discard any existing clots.
        NOTE: Transfer 200 µL of undiluted BM into a 1.5 mL tube for subsequent flow cytometric analysis. Store at 4 °C until use.
      3. Transfer 10 mL BM into a new 50 mL conical tube, add 6 mL of PBS/EDTA (2 mM), 20 mL of human lymphocyte medium, 175 µL of Collagenase B (40 mg/mL), and 175 µL of DNAse I (10 mg/mL). Gently mix the solution and incubate for 30 min at RT on a shaker. Repeat for the remaining BM sample.
    3. Density gradient centrifugation
      1. Prime a 50 mL density gradient centrifugation tube by applying 15 mL of human lymphocyte separating medium into a 50 mL tube. Centrifuge at 1,000 x g for 30 s.
      2. Carefully layer 35 mL of diluted BM on top of the density gradient centrifugation tube filter and centrifuge at 445 x g for 35 min at RT.
        NOTE: Centrifuge settings are low acceleration (3) and no brake (1).
      3. Carefully remove the tube from the centrifuge without shaking.Carefully discard ~20 mL from the upper clear solution without touching the cloudy layer directly on top of the filter.
      4. Carefully transfer the cloudy layer (which is directly on top of the filter and contains the mononuclear cells (MNCs)) into a new 50 mL conical tube and fill up with PBS/EDTA to a final volume of 50 mL.
        NOTE: Combine all MNCs from one BM patient sample into one new tube.
      5. Count the MNCs by transferring 10 µL of the 50 mL cell suspension into a 1.5 mL tube. Add 10 µL of 3% acetic acid with methylene blue. Gently mix and apply 10 µL into a counting chamber. Calculate the number of MNCs.
      6. Centrifuge the MNC suspension at 300 x g for 10 min. Discard the supernatant.
        NOTE: During centrifugation, cool down the centrifuge from RT to 4 °C.
    4. Magnetic selection of CD133+ SCs using human CD133 antibody-linked superparamagnetic iron dextran particles
      NOTE: During this step, work on ice. Store solutions until use at 4 °C. Store MACS permanent magnet, separation columns, and pre-separation filter at 4 °C.
      1. Choose the column size. For <1.2 x 108 MNCs, use MS columns, and for >1.2 x 108 MNCs, use LS columns (see Table of Materials).
      2. To prepare of magnetic selection for 1 x 108 total cell number, carefully resuspend MNCs in 300 µL MACS buffer (4 °C). Add 100 µL FcR blocking reagent (4 °C) and 100 µL CD133 antibody-linked superparamagnetic iron dextran particles (4 °C).
      3. Gently mix the cell suspension and incubate for 30 min at 4 °C. Gently shake the cell suspension during incubation (2–3x).
      4. Add 2 mL of MACS buffer (4 °C) per 1 x 108 total MNCs. Gently mix the cell suspension and centrifuge at 300 x g for 10 min at 4 °C.
      5. Set up the MACS magnet holder and attach the MACS permanent magnet. Install the MACS column and apply the pre-separation filter on top.
      6. Equilibrate the first MACS MS/LS column and pre-separation filter with 0.5 mL (MS) or 3 mL (LS) of MACS buffer (4 °C).
        NOTE: Never allow the MACS columns to dry out after equilibration.
      7. Discard the supernatant and resuspend the obtained pellet in 500 µL of MACS buffer (4 °C) per 1 x 108 total cell amount. Apply the cell suspension into the pre-separation filter.
      8. Wash the MACS column and pre-separation filter three times using, for each wash, 0.5 mL (MS) or 3 mL (LS) of MACS buffer (4 °C).
        NOTE: During the third washing-step, install a second MACS column into the MACS permanent magnet and equilibrate the second MACS MS/LS column with 0.5 mL (MS) or 3 mL (LS) MACS buffer (4 °C).
      9. Discard the pre-separation filter.
      10. Elute the cell fraction directly onto the second MACS column: add 1 mL (MS) or 5 mL (LS) MACS buffer (4 °C) onto the first MACS column and remove the MACS column from the MACS permanent magnet. Immediately transfer the first MACS column above the second MACS column and push the cell suspension through the MACS column using the supplied plunger.
      11. Wash the MACS column three times, using for each wash 0.5 mL (MS) or 3 mL (LS) MACS buffer (4 °C).
      12. Elute the cell fraction from the column by adding 1 mL (MS) or 5 mL (LS) MACS buffer (4 °C) onto the second MACS column and removing the MACS column from the MACS permanent magnet. Immediately transfer the second MACS column above a 1.5 mL tube (MS) or 15 mL conical tube (LS) and push the cell suspension through the MACS column using the supplied plunger.
      13. Centrifuge at 300 x g for 10 min at 4 °C.Carefully discard the supernatant and resuspend the obtained pellet in 100 µL of MACS buffer (4 °C).
      14. Count the CD133+ cells: transfer 6 µL of the cell suspension into a 1.5 mL tube. Add 6 µL trypan blue solution (0.4%). Gently mix and apply 10 µL into a counting chamber. Calculate the number of CD133+ cells.
      15. Store the CD133+ cells on ice until seeding.
  2. Characterization of freshly isolated CD133+ SCs
    NOTE: The following work should be carried out in a room protected from bright light (dim lighting only). Keep tubes, buffers, and antibodies on ice unless stated otherwise.
    1. Preparation of the SCs for flow cytometric analysis
      1. Use two samples (each 10 µL) of BM aliquots and one sample of freshly isolated CD133+ SCs (minimum, 1 x 104 cells) for analysis. Transfer the cells into a 1.5 mL tube. Add 10 µL of FcR blocking reagent and fill up with MACS buffer (4 °C) to a volume of 33 µL.
      2. Add the following antibodies onto the inner side of the respective tube (see Table 1): anti-CD34-fluorescein isothiocyanate (FITC) (clone: AC136), anti-CD133/2-phycoerythrin (PE) (clone: 293C3), isotype control mouse IgG 2b-PE, anti-CD45-allophycocyanin (APC)-H7 (clone: 2D1), and 7-aminoactinomycin (AAD). After all antibodies have been added, shake down the antibodies side of the tube. Gently mix the solution (final volume 50 µL) and incubate for 10 min at 4 °C.
      3. Add 1 mL of 1x red blood cell (RBC) lysis buffer, gently mix the suspension, and incubate on ice 10 min. Centrifuge the suspension at 300 x g for 10 min at 4 °C.
      4. Discard the supernatant and resuspend the obtained pellet in 100 µL PBS (4 °C). Store on ice until flow cytometric analysis.
    2. Flow cytometric analysis of CD133+ SCs
      1. To examine the cell viability and purity of CD133+ SC, use an adapted International Society of Hematotherapy and Graft Engineering (ISHAGE) flow cytometry gating strategy41 (see the representative depiction in Figure 1). Use the following order:
        Step 1: selection of cell population (Figure 1A)
        Step 2: selection of CD45+ cells (Figure 1B)
        Step 3: selection of viable CD45+ cells (Figure 1C)
        Step 4: selection of viable CD45+/CD34+ cells (Figure 1D)
        Step 5: selection of viable CD45+/CD34+/CD133+ cells (Figure 1E)
        Run flow cytometer (see Table of Materials) according to the manufacturer's instructions.
      2. Calculate the cell viability and purity using the following equations:

Cell viability formula, Viability [%] = (viable CD45+ cells / CD45+ cells) × 100, equation.   Equation 1

Purity equation for cell analysis, formula for calculating cell population percentages, CD markers.   Equation 2

  1. Cell culture of freshly isolated CD133+ SCs
    1. Aliquot 100x recombinant human cytokine supplement in 100 µL aliquots and store at -20 °C. Thaw one 100x aliquot before the medium preparation.
    2. Prepare the CD133+ SC culture medium, serum-free hematopoietic cell expansion medium supplemented with recombinant human cytokine supplement (final 1x) and 1% penicillin/streptomycin.
    3. Seed 5 x 104 freshly isolated CD133+ SCs for transfection in a 24-well plate with 500 µL culture medium. Incubate the cells at 37 °C, 5% CO2 and 20% O2.
      NOTE: Seed 5 x 104 freshly isolated CD133+ SCs as a control for flow cytometric analysis.

2. Preparation of Transfection Complexes

NOTE: During this step, keep the entire work space ribonuclease (RNAse)-free using RNAse decontamination solution and use only RNAse-free consumable material.

  1. Preparation of miR
    NOTE: The following work should be carried out in a room protected from bright light (dim lighting only).
    NOTE: For the miR-specimen: use Cyanine 3 dye labeled precursor miR and unlabeled precursor miR.
    1. To prepare the miR stock solution (50 µM), dilute the desired amount of miR in the appropriate volume of nuclease-free water (e.g., dilute 5 nmol of miR in 100 µL water). Gently mix the solution, aliquot the miR stock (5 µL), and store below -20 °C.
  2. Preparation of PEI
    1. Prepare the PEI following standard protocols and note the PEI stock concentration31,42 .
  3. Preparation of MNP
    1. Prepare the MNPs following standard protocols and note the MNP stock concentration31,42.
  4. Preparation of miR/PEI complexes
    1. Prepare 5% glucose solution by diluting D-(+)-glucose in nuclease-free water. Gently mix the solution, aliquot the stock (1.5 mL), and store at 4 °C.
      NOTE: The following work should be carried out in a room protected from bright light (dim lighting only).
    2. Use 20 pmol miR per well of a 24-well plate30. Dilute miR in the 5% glucose solution to a final concentration of 0.25 pmol miR/µL.
    3. Calculate the amount of PEI required based on the miR amount (20 pmol, step 2.3.1) and optimal nitrogen (of PEI) to phosphate (of miR) (N/P) ratio based on the PEI stock concentration (step 2.2.1; here, a ratio of 7.5 was used)30. Dilute the PEI in an equal amount of 5% glucose solution based on the equation:
      N/P ratio equation for gene delivery efficiency optimization; formula involves PEI and miR concentration.
      which rearranges to
      Volume of PEI formula, mathematical equation, used in biochemical calculations for gene transfection.    Equation 3
      where PEI volume is in µL and [PEI] is in mM, and N/P (optimized for this protocol) is 0.75. 0.12 is a conversion factor specific to miR.
    4. Add the pre-diluted PEI into the pre-diluted miR and vortex for 30 s.
    5. Incubate the miR/PEI complex for 30 min at RT.
  5. Preparation of the miR/PEI/MNP complexes
    NOTE: The following work should be carried out in a room protected from bright light (dim lighting only).
    1. While incubating the miR/PEI complexes, prepare the MNPs by sonicating the MNPs for 20 min at 35 kHz in the sonicating water bath at RT to ensure the particles are in suspension and not aggregating.
    2. Calculate the number of required MNPs (3 µg iron/mL) based on the MNP stock solution (step 2.3.1).
    3. Add the sonicated MNPs into the miR/PEI complexes and vortex for 30 s. Incubate the miR/PEI/MNP complexes for 30 min at RT.
  6. Preparation of the transfection complexes for four-color labeling using SIM
    NOTE: Use unlabeled precursor miR.
    1. Label the miR with Cyanine 5 dye using a Cyanine 5 dye miR labeling kit (see Table of Materials) following the manufacturer's instructions. Measure the final miR concentration using the listed spectrophotometer (see Table of Materials) in accordance to the pre-setting provided by the manufacturer (nucleic acid, RNA-40). Aliquot the miR (5 µL) and store at -80 °C protected from light.
    2. Label the PEI with a bright green fluorescence dye using an appropriate protein labeling kit (see Table of Materials) following the manufacturer's instructions. Define the final PEI concentration using the Ninhydrin assay (step 2.2.1). Aliquot the PEI (in the volume calculated based on Equation 3) and store at 4 °C protected from light.
    3. Label the MNPs with rhodamine dye conjugated to biotin using a ratio of 1:1,000 w/w, dye to MNP. Mix the rhodamine dye and MNPs concurrently with the preparation of the miR/PEI complex formation and incubate the solution for 30 min in the dark. Directly use labeled MNPs.

3. Transfection of CD133+ SCs

NOTE: During this step, keep the entire work space ribonuclease (RNAse)-free using RNAse decontamination solution and use only RNAse-free consumable material
NOTE: The following work should be carried out in a room protected from bright light (dim lighting only).

  1. Add the prepared miRNA/PEI/MNP complex dropwise directly into the appropriate well containing the freshly isolated CD133+ SCs in culture medium.
  2. Mix gently by rocking the plate back and forth. Incubate the cells for 18 h at 37 °C, 5% CO2 and 20% O2.

4. Analysis of the miR Transfection

  1. Examination of the miR uptake efficiency and the cytotoxicity of transfection complexes
    NOTE: For the miR-specimen: use Cyanine 3 dye labeled precursor miR for the uptake evaluation and non-transfected CD133+ SCs for the flow cytometry control gates.
    NOTE: The following work should be carried out in a room protected from bright light (dim lighting only).
    NOTE: Keep the tubes, buffers, and antibodies on ice unless stated otherwise.
    1. Prepare the paraformaldehyde stock solution (PFA, 4%) by diluting 4 g PFA in 100 mL PBS and heating the solution to 80 °C. Vigorously mix the solution and adjust the pH value to 7.3. Aliquot the PFA stock (1.5 mL) and store at -20 °C.
    2. 18 h after transfection, collect each well in a separate 1.5 mL tube. Wash each well once with 500 µL of PBS and transfer this cell suspension to the same tube.
    3. Centrifuge at 300 x g for 10 min at 4 °C.Discard the supernatant and resuspend the obtained pellet in 100 µL of 4 °C MACS buffer.
    4. Add 0.5 µL of an amine reactive dye to distinguish between live and dead cells, gently mix the suspension, and incubate for 10 min at 4 °C. Add 1 mL PBS (4 °C) and centrifuge at 300 x g for 10 min at 4 °C.
    5. Discard the supernatant, resuspend the obtained pellet in 100 µL PBS, add 33 µL PFA (4%), and store on ice or at 4 °C until flow cytometric analysis.
    6. Arrange the gating strategy according to the representative depiction in Figure 2. Run the samples on the flow cytometer (see Table of Materials) according to the manufacturer's instructions.
  2. Characterization of the miR-transfected CD133+ SCs
    NOTE: Used unlabeled precursor miR as well as non-transfected CD133+ SCs for flow cytometry control gates.
    1. Collect each well in a separate 1.5 mL tube and centrifuge at 300 x g for 10 min at 4 °C.
    2. Use the gating strategy as described in step 1.2.
  3. Examination of the intracellular distribution of transfection complexes by SIM
    1. Prepare the complexes and transfect the cells as described in Section 2 and Section 3.
    2. 18 h after the transfection, collect each well in a separate 1.5 mL tube. Wash each well once with 500 µL of PBS and transfer the wash into the well's respective tube. Centrifuge at 300 x g for 10 min at 4 °C.
    3. Discard the supernatant and resuspend the obtained pellet in 1 mL of 2% fetal bovine serum (FBS) in PBS, gently mix the suspension, and centrifuge at 300 x g for 10 min at 4 °C.
    4. Discard the supernatant, resuspend the obtained pellet in 100 µL of PBS, and add 33 µL PFA (4%) for 20 min.
    5. Transfer the cell suspension into a new 24-culture plate containing sterile glass coverslips and centrifuge at 300 x g for 10 min at 4 °C.
    6. Discard the supernatant and add 500 µL of PBS. Gently shake the plate and discard PBS.
    7. Place the prepared coverslips on the microscopic slides using aqueous mounting medium to preserve fluorescence and dry them at RT for at least 1 h.
    8. Acquire images using a 100X oil immersion objective. SIM settings: 405, 488, 561, and 633 nm laser lines for excitation and z-stacks with a 16-bit depth at 3 angles, 3 phases, with averaging 4, grids per laser line: 23 µm – 405, 34 µm – 488, 42 µm – 561, 51 µm – 633.

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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

List of materials used in this article
NameCompanyCatalog NumberComments
7-AADBD Biosciences559925
Acetic Acid with Methylene BlueStemcell Technologies70603%
anti-CD133/2-PE (clone: 293C3)Miltenyi Biotec GmbH130-090-853
anti-CD34-FITC (clone: AC136)Miltenyi Biotec GmbH130-081-001
anti-CD45-APC-H7 (clone: 2D1)BD Biosciences560178
rhodamine dye; Atto 565 dye conjugated to biotinATTO-TEC GmbHAD 565-71
BD FACS LSRII flow cytometerBD Biosciences
BD FACSDiva Software 6.1.2BD Biosciences
BSASigma-Aldrich GmbHA7906
CD133 antibody-linked superparamagnetic iron dextran particles; CD133 MicroBead KitMiltenyi Biotec GmbH130-097-049
collagenase BRoche Diagnostics GmbH11088831001
counting chamberPaul Marienfeld GmbH & Co. KG
Cyanine 3 dye labelled precursor miR; Cy3 Dye-Labeled Pre-miR Negative Control #1AmbionAM17120
Cyanine 5 dye miR labelling kit; Cy5 dye Label IT miRNA Labeling KitMirus BioMIR 9650
DNAse IRoche Diagnostics GmbH10104159001(100 U/mL)
ELYRA PS.1 LSM 780 confocal microscopeCarl Zeiss Jena GmbH
FcR Blocking Reagent, humanMiltenyi Biotec GmbH130-059-901
bright green protein labeling kit; Oregon Green 488 Protein Labeling KitThermo Fisher ScientificO10241
aqueous mounting medium; FluoroshieldSigma-Aldrich GmbHF6182
density gradient centrifugation tube; Leukosep Centrifuge TubeGreiner Bio-One89048-932
MACS magnet holder; MACS MultiStandMiltenyi Biotec GmbH130-042-303
MACS pre-separation filterMiltenyi Biotec GmbH130-041-40730 µm
MACS separation column (MS / LS)Miltenyi Biotec GmbH130-042-201 / 130-042-401
MACS permanent magnet; MACS SeparatorMiltenyi Biotec GmbH130-042-302
Millex-HV PVDF FilterMerckSLHV013SL0.45 μm
mouse IgG 2b-PEMiltenyi Biotec GmbH130-092-215
amine reactive dye; Near-IR LIVE/DEAD Fixable Dead Cell Stain KitThermo Fisher ScientificL10119
human lymphocyte separating medium; PancollPan Biotech GmbHP04-60500density: 1.077 g/mL
PBSPan Biotech GmbHP04-53500without Ca and Mg
PEISigma-Aldrich GmbH408727branched; 25 kDa
Penicillin/StreptomycinThermo Fisher Scientific15140122100 U/mL, 100 μg/mL
PFAMerck Schuchardt OHG1040051000
unlabelled precursor miR; Pre-miR miRNA Precursor Negative Control #1AmbionAM17110
RBC lysis buffereBioscience00-4333-57
RNAse decontamination solution; RNaseZapThermo Fisher ScientificAM9780
human lymphocyte medium; Roswell Park Memorial Institute (RPMI) 1640 mediumPan Biotech GmbHP04-16500
recombinant human cytokine supplement; StemSpan CC100Stemcell Technologies2690
serum-free haematopoietic cell expansion medium; StemSpan H3000Stemcell Technologies9800
Streptavidin MagneSphere Paramagnetic ParticlesPromega CorporationZ5481
Trypan Blue solutionSigma-Aldrich GmbHT81540.4 %
UltraPure EDTAThermo Fisher Scientific155750200.5 M; pH 8.0
ZEN2011 softwareCarl Zeiss Jena GmbH
NanoDrop 1000 SpectrophotometerThermo Fisher Scientific
Sonorex RK 100 SH sonicating water bathBandelin electronicUltrasonic nominal output: 80 W; Ultrasonic frequency: 35 kHz

References

  1. 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).
  2. Lee, S., Yoon, Y. -S. Revisiting cardiovascular regeneration with bone marrow-derived angiogenic and vasculogenic cells. British journal of pharmacology. 169 (2), 290-303 (2013).
  3. Beksac, M., Preffer, F. Is it time to revisit our current hematopoietic progenitor cell quantification methods in the clinic? Bone marrow transplantation. 47 (11), 1391-1396 (2012).
  4. Bongiovanni, D., et al. The CD133+ cell as advanced medicinal product for myocardial and limb ischemia. Stem cells and development. 23 (20), 2403-2421 (2014).
  5. Wang, X., et al. The Clinical Status of Stem Cell Therapy for Ischemic Cardiomyopathy. Stem cells international. 2015, 135023(2015).
  6. Ma, N., et al. Intramyocardial delivery of human CD133+ cells in a SCID mouse cryoinjury model: Bone marrow vs. cord blood-derived cells. Cardiovascular research. 71 (1), 158-169 (2006).
  7. Rafii, S., Lyden, D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nature medicine. 9 (6), 702-712 (2003).
  8. Wang, D., Gao, G. State-of-the-art human gene therapy: part I. Gene delivery technologies. Discovery medicine. 18 (97), 67-77 (2014).
  9. Sart, S., Ma, T., Li, Y. Preconditioning stem cells for in vivo delivery. BioResearch open access. 3 (4), 137-149 (2014).
  10. Liu, J., et al. Early stem cell engraftment predicts late cardiac functional recovery: preclinical insights from molecular imaging. Circulation. Cardiovascular imaging. 5 (4), 481-490 (2012).
  11. Lang, C., et al. In vivo comparison of the acute retention of stem cell derivatives and fibroblasts after intramyocardial transplantation in the mouse model. European journal of nuclear medicine and molecular imaging. 41 (12), 2325-2336 (2014).
  12. Goussetis, E., et al. Intracoronary infusion of CD133+ and CD133-CD34+ selected autologous bone marrow progenitor cells in patients with chronic ischemic cardiomyopathy: cell isolation, adherence to the infarcted area, and body distribution. Stem cells. 24 (10), Dayton, Ohio. 2279-2283 (2006).
  13. Caveliers, V., et al. In vivo visualization of 111In labeled CD133+ peripheral blood stem cells after intracoronary administration in patients with chronic ischemic heart disease. Q J Nucl Med Mol Imaging. 51 (1), 61-66 (2007).
  14. Terrovitis, J. V., Smith, R. R., Marbán, E. Assessment and optimization of cell engraftment after transplantation into the heart. Circulation research. 106 (3), 479-494 (2010).
  15. Rosado-de-Castro, P. H., et al. Biodistribution of bone marrow mononuclear cells after intra-arterial or intravenous transplantation in subacute stroke patients. Regenerative medicine. 8 (2), 145-155 (2013).
  16. Kang, W. J., Kang, H. -J., Kim, H. -S., Chung, J. -K., Lee, M. C., Lee, D. S. Tissue distribution of 18F-FDG-labeled peripheral hematopoietic stem cells after intracoronary administration in patients with myocardial infarction. Journal of nuclear medicine official publication, Society of Nuclear Medicine. 47 (8), 1295-1301 (2006).
  17. Blocklet, D., et al. Myocardial homing of nonmobilized peripheral-blood CD34+ cells after intracoronary injection. Stem cells. 24 (2), Dayton, Ohio. 333-336 (2006).
  18. Penicka, M., et al. One-day kinetics of myocardial engraftment after intracoronary injection of bone marrow mononuclear cells in patients with acute and chronic myocardial infarction. Heart (British Cardiac Society). 93 (7), 837-841 (2007).
  19. Schächinger, V., et al. Pilot trial on determinants of progenitor cell recruitment to the infarcted human myocardium. Circulation. 118 (14), 1425-1432 (2008).
  20. Dedobbeleer, C., et al. Myocardial homing and coronary endothelial function after autologous blood CD34+ progenitor cells intracoronary injection in the chronic phase of myocardial infarction. Journal of cardiovascular pharmacology. 53 (6), 480-485 (2009).
  21. Musialek, P., et al. Randomized transcoronary delivery of CD34(+) cells with perfusion versus stop-flow method in patients with recent myocardial infarction: Early cardiac retention of (m)Tc-labeled cells activity. Journal of nuclear cardiology official publication of the American Society of Nuclear Cardiology. 18 (1), 104-116 (2011).
  22. Kyrtatos, P. G., et al. Magnetic tagging increases delivery of circulating progenitors in vascular injury. JACC. Cardiovascular interventions. 2 (8), 794-802 (2009).
  23. Vandergriff, A. C., et al. Magnetic targeting of cardiosphere-derived stem cells with ferumoxytol nanoparticles for treating rats with myocardial infarction. Biomaterials. 35 (30), 8528-8539 (2014).
  24. Huang, Z., et al. Magnetic targeting enhances retrograde cell retention in a rat model of myocardial infarction. Stem cell research & therapy. 4 (6), 149(2013).
  25. Yanai, A., et al. Focused magnetic stem cell targeting to the retina using superparamagnetic iron oxide nanoparticles. Cell transplantation. 21 (6), 1137-1148 (2012).
  26. Arbab, A. S., Jordan, E. K., Wilson, L. B., Yocum, G. T., Lewis, B. K., Frank, J. A. In vivo trafficking and targeted delivery of magnetically labeled stem cells. Human gene therapy. 15 (4), 351-360 (2004).
  27. Cores, J., Caranasos, T. G., Cheng, K. Magnetically Targeted Stem Cell Delivery for Regenerative Medicine. Journal of functional biomaterials. 6 (3), 526-546 (2015).
  28. Cheng, K., et al. Magnetic enhancement of cell retention, engraftment, and functional benefit after intracoronary delivery of cardiac-derived stem cells in a rat model of ischemia/reperfusion. Cell transplant. 21 (6), 1121-1135 (2012).
  29. Li, W., et al. Enhanced thoracic gene delivery by magnetic nanobead-mediated vector. The journal of gene medicine. 10 (8), 897-909 (2008).
  30. Müller, P., et al. Magnet-Bead Based MicroRNA Delivery System to Modify CD133+ Stem Cells. Stem cells international. 2016, 7152761(2016).
  31. Voronina, N., et al. Non-viral magnetic engineering of endothelial cells with microRNA and plasmid-DNA-An optimized targeting approach. Nanomedicine nanotechnology, biology, and medicine. , (2016).
  32. Noort, W. A., et al. Mesenchymal stromal cells to treat cardiovascular disease: strategies to improve survival and therapeutic results. Panminerva Med. 52 (1), 27-40 (2010).
  33. Jakob, P., Landmesser, U. Role of microRNAs in stem/progenitor cells and cardiovascular repair. Cardiovascular Research. 93 (4), 614-622 (2012).
  34. Sen, C. K. MicroRNAs as new maestro conducting the expanding symphony orchestra of regenerative and reparative medicine. Physiological genomics. 43 (10), 517-520 (2011).
  35. Papapetrou, E. P., Zoumbos, N. C., Athanassiadou, A. Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects. Gene therapy. 12, Suppl 1. S118-S130 (2005).
  36. Chira, S., et al. Progresses towards safe and efficient gene therapy vectors. Oncotarget. 6 (31), 30675-30703 (2015).
  37. Hobel, S., Aigner, A. Polyethylenimines for siRNA and miRNA delivery in vivo. Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology. 5 (5), 484-501 (2013).
  38. Villate-Beitia, I., Puras, G., Zarate, J., Agirre, M., Ojeda, E., Pedraz, J. L. First Insights into Non-invasive Administration Routes for Non-viral Gene Therapy. Gene Therapy - Principles and Challenges. Hashad, D. , (2015).
  39. Cubillos-Ruiz, J. R., Sempere, L. F., Conejo-Garcia, J. R. Good things come in small packages: Therapeutic anti-tumor immunity induced by microRNA nanoparticles. Oncoimmunology. 1 (6), 968-970 (2012).
  40. Schade, A., et al. Magnetic nanoparticle based nonviral microRNA delivery into freshly isolated CD105(+) hMSCs. Stem Cells Int. 2014, 197154(2014).
  41. Sutherland, D. R., Anderson, L., Keeney, M., Nayar, R., Chin-Yee, I. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International society of hematotherapy and graft engineering. Journal of hematotherapy. 5 (3), 213-226 (1996).
  42. Voronina, N., et al. Preparation and in vitro characterization of magnetized mir-modified endothelial cells. Journal of visualized experiments. (123), (2017).
  43. Stamm, C., et al. Intramyocardial delivery of CD133+ bone marrow cells and coronary artery bypass grafting for chronic ischemic heart disease:Safety and efficacy studies. The journal of thoracic and cardiovascular surgery. 133 (3), 717-725 (2007).
  44. King, A., et al. REpeated AutoLogous Infusions of STem cells In Cirrhosis (REALISTIC): A multicentre, phase II, open-label, randomised controlled trial of repeated autologous infusions of granulocyte colony-stimulating factor (GCSF) mobilised CD133+ bone marrow stem cells in patients with cirrhosis. A study protocol for a randomised controlled trial. BMJ open. 5 (3), e007700(2015).
  45. Martinez, H. R., et al. Stem cell transplantation in amyotrophic lateral sclerosis patients: methodological approach, safety, and feasibility. Cell transplantation. 21 (9), 1899-1907 (2012).
  46. Jimenez-Quevedo, P., et al. Selected CD133(+) progenitor cells to promote angiogenesis in patients with refractory angina: final results of the PROGENITOR randomized trial. Circulation research. 115 (11), 950-960 (2014).
  47. Raval, A. N., et al. Bilateral administration of autologous CD133+ cells in ambulatory patients with refractory critical limb ischemia: lessons learned from a pilot randomized, double-blind, placebo-controlled trial. Cytotherapy. 16 (12), 1720-1732 (2014).
  48. Andreone, P., et al. Reinfusion of highly purified CD133+ bone marrow-derived stem/progenitor cells in patients with end-stage liver disease: A phase I clinical trial. Digestive and liver disease. 47 (12), 1059-1066 (2015).
  49. Arici, V., et al. Autologous immuno magnetically selected CD133+ stem cells in the treatment of no-option critical limb ischemia: clinical and contrast enhanced ultrasound assessed results in eight patients. Journal of translational medicine. 13, 342(2015).
  50. Zali, A., et al. Intrathecal injection of CD133-positive enriched bone marrow progenitor cells in children with cerebral palsy: feasibility and safety. Cytotherapy. 17 (2), 232-241 (2015).
  51. Al-Zoubi, A., et al. Transplantation of purified autologous leukapheresis-derived CD34+ and CD133+ stem cells for patients with chronic spinal cord injuries: long-term evaluation of safety and efficacy. Cell transplantation. 23, Suppl 1. S25-S34 (2014).
  52. Isidori, A., et al. Positive selection and transplantation of autologous highly purified CD133(+) stem cells in resistant/relapsed chronic lymphocytic leukemia patients results in rapid hematopoietic reconstitution without an adequate leukemic cell purging. Biology of blood and marrow transplantation. 13 (10), 1224-1232 (2007).
  53. Nasseri, B. A., et al. Autologous CD133+ bone marrow cells and bypass grafting for regeneration of ischaemic myocardium: the Cardio133 trial. European heart journal. 35 (19), 1263-1274 (2014).
  54. Steinhoff, G., et al. Cardiac Function Improvement and Bone Marrow Response -: Outcome Analysis of the Randomized PERFECT Phase III Clinical Trial of Intramyocardial CD133(+) Application After Myocardial Infarction. EBioMedicine. 22, 208-224 (2017).
  55. Muller, P., et al. Intramyocardial fate and effect of iron nanoparticles co-injected with MACS(R) purified stem cell products. Biomaterials. 135, 74-84 (2017).
  56. Müller, P., Gaebel, R., Lemcke, H., Steinhoff, G., David, R. Data on the fate of MACS® MicroBeads intramyocardially co-injected with stem cell products. Data in brief. 13, 569-574 (2017).
  57. Skorska, A., et al. GMP-conformant on-site manufacturing of a CD133+ stem cell product for cardiovascular regeneration. Stem cell research & therapy. 8 (1), 33(2017).
  58. Delyagina, E., Li, W., Ma, N., Steinhoff, G. Magnetic targeting strategies in gene delivery. Nanomedicine (Lond). 6 (9), 1593-1604 (2011).
  59. Schade, A., et al. Innovative strategy for microRNA delivery in human mesenchymal stem cells via magnetic nanoparticles. International journal of molecular sciences. 14 (6), 10710-10726 (2013).
  60. Delyagina, E., et al. Improved transfection in human mesenchymal stem cells: Effective intracellular release of pDNA by magnetic polyplexes. Nanomedicine. 9 (7), London, England. 999-1017 (2014).
  61. Yin, H., Kanasty, R. L., Eltoukhy, A. A., Vegas, A. J., Dorkin, J. R., Anderson, D. G. Non-viral vectors for gene-based therapy. Nature reviews. Genetics. 15 (8), 541-555 (2014).
  62. Chen, J., Guo, Z., Tian, H., Chen, X. Production and clinical development of nanoparticles for gene delivery. Molecular therapy. Methods & clinical development. 3, 16023(2016).
  63. Juliano, R. L. The delivery of therapeutic oligonucleotides. Nucleic Acids Research. 44 (14), 6518-6548 (2016).
  64. Soenen, S. J., Rivera-Gil, P., Montenegro, J. -M., Parak, W. J., de Smedt, S. C., Braeckmans, K. Cellular toxicity of inorganic nanoparticles: Common aspects and guidelines for improved nanotoxicity evaluation. Nano today. 6 (5), 446-465 (2011).
  65. Estelrich, J., Sánchez-Martín, M. J., Busquets, M. A. Nanoparticles in magnetic resonance imaging: From simple to dual contrast agents. International journal of nanomedicine. 10, 1727-1741 (2015).

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CD133 Positive CellsBone Marrow IsolationFlow Cytometry AnalysisMagnetic Activated Cell SortingPolymer Nanoparticle TransfectionStructured Illumination MicroscopyCell Viability Assessment
Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting
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