Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells

Abstract

Background: Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts.

Methods: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling.

Results: 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups.

Conclusion: This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming.

Keywords: human fibroblasts; induced pluripotent stem cells; poly(beta-amino ester) nanoparticles; reprogramming.

Figure 1

PBAE synthesis scheme and structure of monomers used for synthesis. Notes: Acrylate monomers (Bx), amino-alcohol monomers (Sy), and end-capping groups (Ez). The number “x” following the acrylate monomers “B” refers to the number of carbons between acrylate groups in the monomer, the number “y” following the amino-alcohol monomers “S” refers to the number of carbons between the amine group and the alcohol group in the sidechain, and the number “z” following the end-capping groups “E” refers to an arbitrary number used to designate a particular end-capping group. Abbreviation: PBAE, poly(beta-amino ester).

Figure 2

High-throughput screening data for gene delivery efficacy of PBAE polymers to IMR90 cells. Luciferase-encoding DNA is delivered and expression is measured as RLU. Ratios 1.05:1, 1.1:1, 1.2:1, 1:1, and 1:1.05 refer to polymerization conditions and wt/wt is the weight ratio of polymer to DNA. Graphs show the mean, n≥4. Notes: Acrylate monomers (Bx), amino-alcohol monomers (Sy), and end-capping groups (Ez). The number “x” following the acrylate monomers “B” refers to the number of carbons between acrylate groups in the monomer, the number “y” following the amino-alcohol monomers “S” refers to the number of carbons between the amine group and the alcohol group in the sidechain, and the number “z” following the end-capping groups “E” refers to an arbitrary number used to designate a particular end-capping group. Abbreviations: PBAE, poly(beta-amino ester); RLU, relative light units; PEI, polyethylenimine.

Figure 3

Flow cytometry data for IMR-90 cells transfected with EGFP using the leading polymers. Transfection efficacy expressed as percent green fluorescent protein-positive live cells (A) following a single dose and (B) after three doses of B4S4E7 (68%). Graphs show the mean ± standard error of the mean, n≥5. (C) Fluorescence microscopy images of untreated (left) and transfected (right) IMR-90 fibroblasts, showing good viability in both cases. Notes: Acrylate monomers (Bx), amino-alcohol monomers (Sy), and end-capping groups (Ez). The number “x” following the acrylate monomers “B” refers to the number of carbons between acrylate groups in the monomer, the number “y” following the amino-alcohol monomers “S” refers to the number of carbons between the amine group and the alcohol group in the sidechain, and the number “z” following the end-capping groups “E” refers to an arbitrary number used to designate a particular end-capping group. Abbreviation: EGFP, enhanced green fluorescent protein.

Figure 4

Cell viability data for IMR-90 cells transfected with EGFP/PBAE nanoparticles. Graphs show the mean ± standard error of the mean, n≥3. Notes: Acrylate monomers (Bx), amino-alcohol monomers (Sy), and end-capping groups (Ez). The number “x” following the acrylate monomers “B” refers to the number of carbons between acrylate groups in the monomer, the number “y” following the amino-alcohol monomers “S” refers to the number of carbons between the amine group and the alcohol group in the sidechain, and the number “z” following the end-capping groups “E” refers to an arbitrary number used to designate a particular end-capping group. Abbreviations: EGFP, enhanced green fluorescent protein; PBAE, poly(beta-amino ester).

Figure 5

(A) Particle sizing data for PBAE nanoparticles complexed with EGFP plasmid (∼5 kb) and episomal plasmid (∼16 kb). Graph shows the mean ± standard error of the mean, n≥3. (B) Zeta potential data for EGFP/B4S4E7 nanoparticles. Graphs show the mean ± standard error of the mean, n=5. (C) Gel retardation assay data for EGFP/B4S4E7 nanoparticles. Notes: Acrylate monomers (Bx), amino-alcohol monomers (Sy), and end-capping groups (Ez). The number “x” following the acrylate monomers “B” refers to the number of carbons between acrylate groups in the monomer, the number “y” following the amino-alcohol monomers “S” refers to the number of carbons between the amine group and the alcohol group in the sidechain, and the number “z” following the end-capping groups “E” refers to an arbitrary number used to designate a particular end-capping group. Abbreviations: EGFP, enhanced green fluorescent protein; PBAE, poly(beta-amino ester); PBS, phosphate buffered saline; NaAc, sodium acetate.

Figure 6

(A) Dose response curve for fibroblasts transfected with EGFP/B4S4E7 nanoparticles. (B) qPCR analysis data showing EGFP messenger (m)RNA expression levels in RP patient-specific adult dermal fibroblasts transfected with B4S4E7/EGFP nanoparticles. Graph shows the mean ± standard deviation, n=3. (C) qPCR analysis data showing Oct4 mRNA expression levels in RP patient-specific adult dermal fibroblasts transfected with B4S4E7/T3 nanoparticles. Graph shows the mean ± standard deviation, n=3. Notes: Acrylate monomers (Bx), amino-alcohol monomers (Sy), and end-capping groups (Ez). The number “x” following the acrylate monomers “B” refers to the number of carbons between acrylate groups in the monomer, the number “y” following the amino-alcohol monomers “S” refers to the number of carbons between the amine group and the alcohol group in the sidechain, and the number “z” following the end-capping groups “E” refers to an arbitrary number used to designate a particular end-capping group. Abbreviations: EGFP, enhanced green fluorescent protein; qPCR, quantitative polymerase chain reaction; RP, retinitis pigmentosa; T3, three reprogramming plasmids.

Figure 7

(A) Flow cytometry data for IMR-90 cells electroporated with EGFP-N1 plasmid at different voltages, ie, 1,500 V, 1,600 V, and 1,700 V. (B) Fluorescence microscopy image of IMR-90 cells electroporated with EGFP-N1 plasmid at 1,500 V. Abbreviation: EGFP, enhanced green fluorescent protein.

Figure 8

(A) PBAE-nanoparticle based feeder-independent reprogramming strategy. (B) Electroporation based feeder-independent reprogramming strategy. Notes: The green bar represents days. Red arrows show the day of polymer-based transfection; blue arrows show the day of electroporation. Abbreviations: PBAE, poly(beta-amino ester); MEM, minimum essential medium; FBS, fetal bovine serum; bFGF, basic fibroblast growth factor; hiPSC, human induced pluripotent stem cell; NaB, sodium butyrate.

Figure 9

Characterization of EP1-hiPSC-like cell line generated by electroporation of nonintegrating episomal plasmids. Normal morphology is observed at low (A) and high (B) magnification. A normal karyotype is seen in (C). Immunolabeling with markers for pluripotency are shown (D–G). Abbreviation: hiPSC, human induced pluripotent stem cells; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 10

Characterization of EP2-iPSC-like cell line generated by PBAE nanoparticles. Normal morphology is observed in (A) pre-purified and (B) post-purified iPSC-like cells. An abnormal karyotype is seen in (C) (arrows). Immunolabeling with pluripotency markers is shown (D–F). Partial neuronal differentiation of EP2-iPSC-like cell line with regional variation (G and H). Abbreviations: iPSC, induced pluripotent stem cells; PBAE, poly(beta-amino ester); DAPI, 4′,6-diamidino-2-phenylindole.

Figure 11

Neuronal differentiation of EP1-iPSC-like cell line generated by electroporation (A). Neural rosettes (B) (indicated by yellow circle) and mature neurons (C) low and (D) high magnification can be generated from these cells. Immunostaining with markers for neuronal lineages, Pax6 and Tuj1, are shown in (E), (F) low, and (G) high magnifications. Retinal structures including pseudostratified neuroepithelial optic cups (H) and retinal pigment epithelium low (I) and high (J) magnifications can also be generated. Abbreviation: iPSC, induced pluripotent stem cells.

Figure 12

Fluorescence microscopy images (10×) of hiPSC-like cells transfected with a single dose of B4S4E7/EGFP nanoparticles. Images show healthy morphology post-transfection (A1), an edge effect in larger colonies (A2), and middle-transfected + edge-transfected cells in smaller colonies (A3). Image (20×) of hiPSC-like colony transfected with (B) two doses of B4S4E7 and EGFP complexes and (C) one dose with B4S4E7 and PiggyBac 2 plasmid system complexes. Notes: Acrylate monomers (Bx), amino-alcohol monomers (Sy), and end-capping groups (Ez). The number “x” following the acrylate monomers “B” refers to the number of carbons between acrylate groups in the monomer, the number “y” following the amino-alcohol monomers “S” refers to the number of carbons between the amine group and the alcohol group in the sidechain, and the number “z” following the end-capping groups “E” refers to an arbitrary number used to designate a particular end-capping group. Abbreviations: hiPSC, human induced pluripotent stem cells; EGFP, enhanced green fluorescent protein.