1Department of Molecular Carcinogenesis and Center for Cancer Epigenetics, University of Texas M.D. Anderson Cancer Center, 2Department of Cell Biology, Poznan University of Medical Sciences, 3Department of Molecular Biology, The Scripps Research Institute
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Polak, U., Hirsch, C., Ku, S., Gottesfeld, J., Dent, S. Y. R., Napierala, M. Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts. J. Vis. Exp. (60), e3416, doi:10.3791/3416 (2012).
Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.
1. Virus production and transduction
3. Isolation of hiPSC colonies
4. Representative Results
Efficient transduction with retrovirus-containing media is critical for successful reprogramming. It is recommended to conduct the entire transfection/infection procedure using a GFP expressing virus every single reprogramming experiment to monitor the efficiency as shown in Figure 1. The titer of the GFP expressing virus determined, as described in 10, through the transduction of human fibroblasts using non-concentrated viral media was typically in the range of 0.5 - 5 x 107 viral particles per ml (vp/ml).
Fibroblasts change morphology as early as 2 days after the last infection. Trypsinized human fibroblasts should be carefully counted prior to seeding them on the MEF feeder cells. It is recommended to seed cells at 3 different densities (6x103, 1.2x104, 2.5x104 per single well of a 6 well plate) since each cell line demonstrates different growth characteristic and seeding density is critical for the efficiency of reprogramming. Sodium butyrate used for the initial 7 - 14 days of reprogramming increases the efficiency of hiPSC formation approximately 5 fold. Frequently, especially when infected fibroblasts were seeded at higher density, the fibroblast-like cells can overgrow a culture dish and cover hiPSC colonies as shown in Figure 2A. In this case, the fibroblast layer can be carefully lifted and removed to uncover hiPSC colonies (Figure 2B). Subsequently, hiPSC colonies should be rinsed with hES media and stained with Tra-1-81 antibody. Depending on the fibroblast cells, approximately 20 - 40% of colonies demonstrating with iPSC-like morphology do not stain with Tra-1-81 antibody. Identified hiPSC colonies can be transferred from a plate within next 12 - 24h. Prolonged incubation will result in rapid differentiation of hiPSCs. Colonies can be manually passaged onto either MEF feeder cells or Matrigel-coated plates. After expansion established hiPSC clones should be tested using immunocytochemistry (ICC) for the expression of pluripotency markers as demonstrated in Figure 3. Additionally, a detailed molecular characterization of the generated iPS cell lines should include: analyses of the pluripotency gene expression using RT-PCR, demonstration of the DNA demethylation at the promoters of pluripotency genes and analyses of the transgenes silencing 9.
Figure 1. The efficiency of viral transduction determined using GFP expressing retrovirus. (A, B) Human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository) were visualized after two consecutive infections with GFP retroviral media. (C, D) Human fibroblasts were infected with the same batch of the GFP retroviral media followed by centrifugation of the cells directly on the 6-well plates for 1h at 1600g. Images were captured 48h after infection.
Figure 2. Identification and isolation of hiPSC colonies. (A) Phase contrast image of a plate containing hiPSCs surrounded by fibroblasts. The cells were cultured for 21 days on hES media. (B, C) The same hiPSC colony after removal of the surrounding fibroblast layer. (D) Correctly reprogrammed hiPSC colonies are identified by live staining with Tra-1-81 surface marker antibody, cut using a sterile needle (E, F) and transferred to separate wells of a 24-well plate.
Figure 3. Expression of the pluripotency specific markers Oct3/4, Nanog, Sox2, SSEA4 and Tra-1-60 in hiPSCs was determined by immunocytochemistry.
Studying human diseases, especially neurological and neurodegenerative, has been particularly challenging due to the inaccessibility of adequate human cellular models. The ability to reprogram easily obtainable somatic cells into induced pluripotent stem cells and the potential to differentiate them into diverse cell types opened a possibility to create cellular models of genetic diseases. In addition, iPSCs hold a great promise in the future of regenerative medicine. Therefore, it is essential to develop and optimize reliable, efficient and safe methods of reprogramming somatic cells to pluripotency.
From the perspective of therapeutic applications, it is crucial to develop safe approaches of iPSC generation lacking footprint mutations in the host genome. Methods of somatic cell reprogramming without introducing permanent alterations into the genome of reprogrammed cells such as transfection of episomal vectors, use of excisable transposons, adenoviral infection, and direct delivery of mRNA or proteins have already been established 11-15. Thus far the reprogramming efficiency using these transgene-free methods is low and depends on the character, type and age of reprogrammed somatic cells. Therefore, these protocols are not suitable for reprogramming late passage, adult somatic cells frequently deposited in cell repositories. Additionally, to enable detailed characterization of multiple iPSC lines and to establish new models of human diseases and to conduct high-throughput drug screens, reprogramming of somatic cells using viral delivery of transcription factors is an adequate strategy. To date more than 20 studies reported generation of patient-specific iPSCs to model human neurological and neuromuscular diseases. In all but one cases the iPSCs were generated using lentiviral or retroviral transduction 16.
Our data demonstrate that a simple protocol based on the retroviral delivery of OSKM transcription factors can be used to efficiently reprogram adult human fibroblasts, even of a high passage. The amount of virus obtained from a single transfection of Phoenix cells on 10 cm plate is sufficient for more than 10 reprogramming experiments. There are three critical steps in our reprogramming protocol of adult fibroblasts: (i) highly efficient viral transduction facilitated by an additional centrifugation step during transduction which dramatically increases the efficiency of transduction; (ii) seeding density of the transduced fibroblast onto the MEFs and (iii) use of live staining with the Tra-1-81 marker antibody to facilitate selection of potentially fully reprogrammed colonies which can be directly transferred to feeder-free cultures. The efficiency of reprogramming is significantly enhanced by using sodium butyrate during second week of the reprogramming. Moreover, we observed that a greater fraction of the iPSC colonies cultured in the presence of the drug can be expanded and established into fully reprogrammed iPSC lines.
The authors have nothing to disclose.
This work was supported by Friedreich’s Ataxia Research Alliance and a pilot grant from Arnold Family Foundation and The Center for Stem Cells and Developmental Biology at M.D. Anderson Cancer Center.
|Oct3/4 antibody||Santa Cruz Biotechnology, Inc.||sc-8628|
|Nanog antibody||Cell Signaling Technology||4903S|
|Tra-1-60 antibody||EMD Millipore||MAB4360|
|Sox2 antibody||Cell Signaling Technology||3579S|
|Objective marker||Nikon Instruments||MBW10010|
|mTeSR1||Stem Cell Technologies||05850|
|Fugene 6||Roche Group||11814443001|
|Object marker||Nikon Instruments||MBW10010|