Research ArticleHEALTH AND MEDICINE

Engineering human megakaryocytic microparticles for targeted delivery of nucleic acids to hematopoietic stem and progenitor cells

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Science Advances  07 Nov 2018:
Vol. 4, no. 11, eaau6762
DOI: 10.1126/sciadv.aau6762
  • Fig. 1 Loading of pDNA into CMPs or MkMPs through electroporation.

    Loading of Cy5-labeled pGFPns was performed via electroporation under (A) different ratios of pDNA copy number to MP number and (B) different electroporation voltages. MPs were washed with phosphate-buffered saline (PBS) after electroporation. (A and B) The percent of Cy5+ MPs was measured via flow cytometry. To measure the amount of loaded pDNA, MPs were lysed and pDNAs were purified for quantification to calculate the number of loaded pDNA per Cy5+ MP. Loading of pGFPns into CMPs was further verified by (C) PCR amplification of a portion of the enhanced GFP (eGFP) sequence and (D) super-resolution microscopy. CMPs were first stained with PKH26 (red) for recognition. Cyan represents Cy5-pDNA. (E to G) Similarly, MkMPs were loaded with Cy5-labeled pmaxGFP via electroporation under different electroporation voltages at a pDNA/MkMP ratio of 150 × 106. (E) The percent of Cy5+ MP, number of loaded pmaxGFP per Cy5+ MP, and (F) mean fluorescence intensity (MFI) of Cy5+ MPs were measured via flow cytometry. (G) Loading of pmaxGFP into MkMPs was further verified by PCR amplification of a portion of the CMV promoter sequence. Error bars in (A), (B), (E), and (F) represent SEM of three replicates.

  • Fig. 2 Delivery of pDNA to HSPCs via MPs.

    (A) Schematic showing the separation of aggregates from individual MPs after loading of pDNA via electroporation. (B) Loading of pGFPns was confirmed in day 1 cells from the coculture of HSPCs with pGFPns-loaded CMPs via PCR amplification of a portion of the eGFP sequence. (C) Schematic of FT setup with 0.4-μm membrane insert. HSPCs were first seeded onto the membrane for 30 min, and MPs were then added to the culture. (D and E) Day 1 HSPCs were cocultured with Cy5-pGFPns–loaded MPs in Eppendorf tubes (Tube), the FT system (FT), the FT system with centrifugation (FT + Centri.), or the FT system but with medium supplemented with PB (8 μg/ml) (FT + PB). Cells were harvested for (D) flow cytometric analysis of Cy5 signal at 24 hours and (E) eGFP-mRNA and GAPDH-mRNA quantification by qRT-PCR at 72 hours. (F and G) Day 1 HSPCs were cocultured with pmaxGFP-loaded (F) CMPs or (G) MkMPs under five coculture conditions (Tube, FT, FT + Centri., FT + PB, and FT + PB + Centri.). Cells were harvested at 24, 48, and 72 hours for measuring GFP expression by flow cytometry. Native MPs were used as control (Blank MP). Data in (D) and (E) represent averages of three biological replicates ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 HSPCs cocultured with Cy5-pGFPns–loaded MPs were harvested and fixed at 24 and 72 hours.

    The location of Cy5-pGFPns (cyan) and eGFP expression (green) were examined by super-resolution microscopy. Nucleus was stained with DAPI (blue), and cellular membrane was stained with DiI (red).

  • Fig. 4 Examining the delivery of pDNA to HSPC nucleus via nuclei analysis.

    Day 1 HSPCs were cocultured with Cy5-pmaxGFP–loaded (A and C) CMPs or (B and D) MkMPs in Eppendorf tube (Tube), FT system with centrifugation (FT + Centri.), FT supplemented with PB (8 μg/ml) (FT + PB), or the combination of PB with centrifugation in FT system (FT + PB + Centri.). Both cells and isolated nuclei were harvested for flow cytometric analysis on (A and B) Cy5+ nuclei percentage or (C and D) MFI of Cy5+ nuclei at 24 hours. Data represent averages of three biological replicates ± SEM.

  • Fig. 5 Silencing of c-myb through MkMP delivery of siRNA to HSPCs enhanced megakaryocytic differentiation.

    MkMPs were loaded with Alexa 647–labeled siRNA (green), siRNA negative control (siR-Neg, gray), or siRNA targeting MYB (siR-MYB, yellow) by electroporation. HSPCs were cocultured with siRNA-loaded MkMPs, MkMPs without any siRNA (MkMP control, orange), or without MkMPs (No MkMP, blue). (A) Delivery efficiency of siRNA to HSPC via MkMPs was examined on the basis of Alexa 647+ percentage of cells by flow cytometric analysis. (B and C) Cells from each coculture were harvested for flow cytometric analysis on CD41+ (%) at days 3, 5, and 8 and on CD34CD41+ (%) at day 8. (D) Level of c-myb was quantified by qRT-PCR from each coculture at 24 hours, normalized to the expression of GAPDH as a reference gene. Data in (B) to (D) represent averages of three biological replicates ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 Functional delivery of miR-486-5P to HSPCs via MkMPs enhanced megakaryocytic differentiation.

    MkMPs were loaded with 8 μM miR-NC or miR-486-5P mimics by electroporation. CD34+ HSPCs (60,000) were cocultured with vehicle control (blue, circle), MkMP control (orange, triangle), miR-NC–loaded MkMPs (gray, square), or miR-486-5P–loaded MkMPs (gold, diamond) for up to 8 days. (Top) A number of total cells or Mks were counted at day 8 of coculture. (Bottom) CD41 (Mk marker) expression was examined at days 3, 5, and 8 of coculture by flow cytometric analysis. Three biological replicates of coculture are shown.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/11/eaau6762/DC1

    Fig. S1. Physical and functional characterization of CMP.

    Fig. S2. The effect of electroporation temperature on pDNA loading efficiency.

    Fig. S3. Flow cytometric histograms.

    Table S1. Primers for amplification in PCR and qRT-PCR.

    Table S2. Estimation of DNA copies in loaded EVs from the literature.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Physical and functional characterization of CMP.
    • Fig. S2. The effect of electroporation temperature on pDNA loading efficiency.
    • Fig. S3. Flow cytometric histograms.
    • Table S1. Primers for amplification in PCR and qRT-PCR.
    • Table S2. Estimation of DNA copies in loaded EVs from the literature.

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