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Generation of Human Induced Pluripotent Stem Cells by Simple Transient Transfection of Plasmid DNA Encoding Reprogramming Factors

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Generation of Human Induced Pluripotent Stem Cells by Simple Transient Transfection of Plasmid DNA Encoding Reprogramming Factors

Karim Si-Tayeb et al. BMC Dev Biol.

Abstract

Background: The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS) cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry.

Results: Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture.

Conclusions: Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.

Figures

Figure 1
Figure 1
Overview of approach used to generate iPS cells by transient transfection of plasmid DNA. A) Schematic and timeline of the protocol used to generate human iPS cells by sequential transient transfection of plasmids expressing OCT4, NANOG, SOX2, and LIN28 cDNAs. B) Photographs showing colonies expressing alkaline phosphatase activity after lentivirus-based reprogramming (left) or plasmid transfection-based reprogramming (right) of human fibroblasts.
Figure 2
Figure 2
Analysis of pluripotency of iPS cells generated by plasmid transfection. A) Micrographs comparing the morphology of the plasmid-derived iPS cells (iPSK3) and human ES cell line H9.14 cultured on mitotically inactivated MEFs and alkaline phosphatase activity identified by histochemistry (Scale bar = 100 μm). B) Immunostaining revealing the presence of OCT4 (red) and SSEA4 (green) in plasmid-derived iPSK3 cells and control H9 human ES cells. Cell nuclei were detected using DAPI stain (Blue) (Scale bar = 100 μm). C) Representative FACS analysis demonstrating that ≥ 99% of iPSK3 cells in culture (passage 8-10) express markers of pluripotency including OCT4, SSEA4, TRA1-60 and TRA1-81. Cells expressing the fibroblast marker CD13 were not detected. D) Micrographs of H & E stained sections through teratomas that formed from iPSK3 cells after injection into immune deficient mice. Cell types derived from all three germ layers - ectoderm, endoderm and mesoderm (indicated with *) - were detected (Scale bar = 100 μm). E) Karyotype of iPSK3 cells revealed a normal distribution of 46 chromosomes with XY sex chromosomes. Chromosomal rearrangements were not detected by G-banding. F) STR analyses using CODIS primers demonstrated that the DNA fingerprint of iPSK3 cells was indistinguishable from that of CRL2097 foreskin fibroblasts used as recipients for reprogramming.
Figure 3
Figure 3
Human iPSK3 cells are devoid of plasmid DNA. A) Southern blot analysis of genomic DNA from control iPS cells, iPSC6 and iPSC2 [14], generated by lentivirus transduction, human ES cells (H9, H9.14, and H9.15), or iPSK3 cells. DNA was digested with BamH1 and blots were hybridized with probes to detect FOXD3 as a loading control (left) or the puromycin N-acetyl transferase gene (right), which is present in both plasmid and lentiviral vectors. B) PCR analysis on total DNA extracted from either human ES cells (H9, H9.14 and H9.15), which lack exogenous DNA sequences, iPSC2 cells which contain integrated viral sequences, and iPSK3 cells using primers that specifically recognize the puromycin N-acetyl-transferase gene. PCR amplification without template DNA was performed to exclude DNA contamination. Primers that specifically recognize a human Alu sequence were used for loading control.
Figure 4
Figure 4
Differentiation of iPSK3 into hepatocyte-like cells. A) Micrograph showing the epithelial-like organization of iPSK3 upon hepatic differentiation. Higher resolution (inset) showing a binucleated cell. Scale bar = 100 μm. B) The hepatocyte-like cells derived from iPSK3 metabolized and transported 5-(and 6)-carboxy-2'-7'-dichlorofluorescein diacetate between cells (high-resolution inset, white arrow), indicating the presence of functional bile transporters. C) Immunocytochemistry revealed the presence of proteins commonly associated with hepatocytes including, FoxA2, Hnf4α, E-cadherin, α-fetoprotein and Albumin. Scale bar = 100 μm.
Figure 5
Figure 5
Differentiation of iPSK3 cells into cardiac cells. A) Immunocytochemistry revealed that clusters of contracting cells expressed cardiac markers including GATA4 in the cells nuclei, detected by DAPI staining, and cardiac-specific Troponin T. High-resolution images revealed a striated pattern of Troponin T staining as expected for a protein associated with myofibrils (Scale bar = 100 μm). B) Intracellular calcium concentration measurements using the fluo-4 AM fluorescent indicator identified rhythmic oscillation correlating with cardiac cell contraction (0.7s between each picture; see movie S2 in supplemental data). C) Contracting cells within the cluster exhibit both an atrial-like calcium transient (cell 1 and 2, lower left panel) and a ventricular-like calcium transient (cell 3 and 4, lower right panel).

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