A nonviral minicircle vector for deriving human iPS cells

Journal: Nature Methods

Publish Date: Feb 7, 2010

Authors: Fangjun Jia1, Kitch

ener D Wilson1,2, Ning Sun1,

Deepak M Gupta3, Mei Huang1, Zongjin Li1,

Nicholas J Panetta3, Zhi Ying Chen4, Robert C Robbins5,

Mark A Kay4 , Michael T Longaker3,6 & Joseph C Wu 1,6

1Departments of Medicine and Radiology, 2Department of Bioengineering, 3Department of Surgery, 4Departments of Pediatrics and Genetics, 5Department of C

ardiothoracic Surgery and 6Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.

Summary:

In this work, the authors demonstrate a new technique to reprogram human adipose stem cells (hASCs) into Induced Pluoripote

nt Stem Cells (iPSCs) with the use of a plasmid like "minicircle DNA" vector. The importance of this work is a demonstration of using none viral vectors to reprogram cells. Reprogramming by viral vectors (pioneered by Shinya Yamanaka), may be carcinogenic since the host genome is altered by the insertion of viral vectors, thus, therapeutic applications are limited. The authors show that the minicircle DNA vector generates the protein transcription factors for reprogramming but does not integrate into the host genome itself, which implies that this method may be a safer technique for therapeutic applications.

The minicircle DNA vectors are similar to plasmids, in this case they are GFP tagged, however they do not have a bacteria replication origin and antibiotic resistance gene. Once electroporated into hASCs, four reprograming t

ranscription factors (Nanog, Oct4, Sox2, Lin 28) and a GFP signal can be observed. Since these minicircle vectors are do not replicate itself in the cell, they are diluted once cells divide.

qPCR, RT-PCR,Southern Blotting, was done to show the generation of reprogramming transcription factors. Bisulfite pyrosequencing was used to confirm a low methylation state. Imunostaining was performed to confirm stem cell markers, and morphology was also confirmed to be similar to iPSCs.

Critique:

Currently iPSCs generation efficiency with Yamanaka viral vectors are approximately 0.01%, however, with this technique, the efficiency is only at around 0.005%. I believe one of the major reasons for this low efficiency is the minicircle delivery method- electroporation. Although electroporation can deliver the vectors effectively, it lowers the viability of the cells greatly. This is due to the lack of control on the electrical field environment around each cell.

Since reprograming needs a continuous dose of the minicircle DNA (for the generation of the transcription vectors), they choose to use lipofectamin for subsequent delivery of the minicircle DNAs, which may have caused low

er delivery efficiency. Thus, i believe, if there was a novel minicircle delivery system which has higher delivery efficiency for the minicircle vectors, it would be a major factor that may be able to boost the overall reprogramming efficiency.

Figures in the paper.

Fig. 1-a=> shows GFP decreases as cell divides, and transcription factors are generated.

Fig. 1-b,c,d=> shows stem cell markers during immunostaining

Fig. 1-e=> RT PCR shows reprogramming genes being active.

Fig. 1-f=> methylation is reduced after transfection.

Fig. 1-g=> heat map comparison

Fig. 1-h=> southern staining

Figure 1 | Generation of iPSCs with minicircle vector. (a) FACS and qPCR analysis of hASCs after transfection with the minicircle vector on day 0. Percentage of GFP positive cells (left) and transcript fold change (right) are plotted. Error bars, s.d. (n = 3). (b) Brightfield (left) and fluorescence (right) images of a day 18 cluster of minicircle-derived iPSCs (mc-iPSCs). (c,d) mc-iPSCs stained for alkaline phosphatase (c) and immunostained for pluripotency markers (d). Scale bars, 100 ␣m (b,d) and 500 ␣m (c). (e) Reverse transcription–PCR (RT-PCR) analysis of three iPSC subclones (iPSC-1s–3s) derived from three separate donors; H9 hESC line; hASCs; and 293-MC negative control (293FT cells 24 h after transfection with the minicircle vector). Endogenous (endo) OCT4, SOX2, NANOG and LIN28 were analyzed. RN18S1 is 18S RNA. (f) Bisulfite pyrosequencing measuring methylation in the promoter regions of OCT4 and NANOG in the indicated cells. Distances upstream of transcription start sites (TSS) are indicated in base pairs. (g) Heatmap of microarray data (top) and scatter plots depicting gene expression fold changes between paired cell types (bottom; the iPSC data are the average of subclones 1 and 2). Highlighted are OCT4, SOX2 and NANOG expression (arrows). Green lines indicate fivefold changes in expression between samples. (h) Southern blot analysis of genomic DNA from the indicated cell lines using probes for OCT4 and SOX2. Lenti-iPSC, lentivirally reprogrammed iPSCs as positive control for genomic integration. MC-EcoRI, minicircle vector after digestion with EcoRI; MC-undigested, undigested minicircle vector.

Figure 2 | Pluripotency of mc-iPSCs. (a) Reverse transcription–PCR (RT-PCR) analysis of undifferentiated (U) and differentiated (D) mc-iPSCs for pluripotency markers (OCT4, NANOG and REX1) and various differentiation markers for the three germ layers (ectoderm, SOX1 and PAX6; mesoderm, T and KDR; endoderm, SOX17 and FOXA2. (b) Phase- contrast images showing cell types differentiated from mc-iPSCs. (c) Subcutaneous injection of mc-iPSCs caused teratomas in severe combined immunodeficient (SCID) mice. Teratomas consisted of all three embryonic germ layers, including neural tissue (neuro.; ectoderm), cartilage (mesoderm) and glandular structures (endoderm). Representative tissue sections from subclone mc-iPSC-1s are shown. Scale bars, 500 ␣m (b) and 100 ␣m (c).