Induction of pluripotency in bone marrow mononuclear cells via polyketal nanoparticle-mediated delivery of mature microRNAs

Abstract

Since the successful generation of induced pluripotent stem cells (iPSC) from adult somatic cells using integrating-viral methods, various methods have been tried for iPSC generation using non-viral and non-integrating technique for clinical applications. Recently, various non-viral approaches such as protein, mRNA, microRNA, and small molecule transduction were developed to avoid genomic integration and generate stem cell-like cells from mouse and human fibroblasts. Despite these successes, there has been no successful generation of iPSC from bone marrow (BM)-derived hematopoietic cells derived using non-viral methods to date. Previous reports demonstrate the ability of polymeric micro and nanoparticles made from polyketals to deliver various molecules to macrophages. MicroRNA-loaded nanoparticles were created using the polyketal polymer PK3 (PK3-miR) and delivered to somatic cells for 6 days, resulting in the formation of colonies. Isolated cells from these colonies were assayed and substantial induction of the pluripotency markers Oct4, Sox2, and Nanog were detected. Moreover, colonies transferred to feeder layers also stained positive for pluripotency markers including SSEA-1. Here, we demonstrate successful activation of pluripotency-associated genes in mouse BM-mononuclear cells using embryonic stem cell (ESC)-specific microRNAs encapsulated in the acid sensitive polyketal PK3. These reprogramming results demonstrate that a polyketal-microRNA delivery vehicle can be used to generate various reprogrammed cells without permanent genetic manipulation in an efficient manner.

Fig. 1

Preparation and characterization of PK3-miRNA particles. A. Pool of four miRNAs was first complexed with the cationic lipid DOTAP. Next, the miRNA:DOTAP complexes were added to an organic solution containing the PK3 polyketal and via a single emulsion/solvent evaporation procedure, spherical particles of submicron size encapsulating miRNA were generated as shown by the scanning electron micrograph. B. The particle size distribution calculated from electron micrograph images shows PK3-miRNA particles ranging from 0.2 to 1 μm, averaging 500 ± 175 nm.

Fig. 2

Release kinetics of PK3-miR (finely ground powder) at pH 5.0, and 7.4 (37 °C). Released miRNAs were measured by qRT-PCR of miR-302a at indicated time points.

Fig. 3

The PK3-miRNA reprogramming protocol. A. Induction of ESC-like cells from Oct4-GFP BM-MNC using PK3-miR particle. Time course demonstrates the culture schedule, treatment for induction of pluripotency, and check points. Oct4-GFP mouse BM-MNC were isolated and cultured for five days followed by treatment with M-CSF and cultured two more days in DMEM. Then the cells were treated with 20 nM of miRNAs (miR-302s and -367) 4 times every other day as indicated and cultured under mouse ESC media. At day 8, Oct4-GFP-positive, ESC-like colonies emerged and were subjected to qRT-PCR for determination of pluripotency genes. At day 14, colonies transferred on to MEF feeders. B. Flow cytometry results of M-CSF treated BM-MNCs showing CD11b, suggesting a macrophage phenotype.

Fig. 4

Detection of Oct4-GFP signal from PK3-miR treated ESC-like mouse colonies. A. Mock-PK3 treated control at day 16. B and C. Expression of GFP signals in the PK3-miR treated cells on feeder cells, which form ESC-like colonies. Pictures taken at day 16 (B) and day 17 (C).

Fig. 5

Morphologies and expression of SSEA-1 of PK3-miR-induced cell colonies. Two representative types of colonies (A and B) were shown which were grown in different culture plates. A colony shown in A is at passage 0 and appeared less mature than the one shown in B which is taken at passage 1. The green fluorescence indicates Oct4-GFP expression and the red fluorescence SSEA-1 staining.

Fig. 6

mRNA expression of pluripotency-related genes in mouse BM-MNCs treated with PK3-miR particle. Uncultured BM-MNCs were used as a negative control. OSKM plasmid was used as a non-viral method control. Mouse ESC was used as a positive control. Data are Mean ± SEM (n = 3), *P < 0.05, **P < 0.01 v.s. BM-MNC.