This work was supported by Friedreich&#x2019;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.

1. Virus production and transduction
<ol>
	<li>Plate Phoenix Ampho cells at a density of ~7-8x106 per 10 cm plate in 10 ml of DMEM medium (DMEM high glucose, 10% FBS heat inactivated, 2 mM L-glutamine, no antibiotics). Place in the incubator and culture overnight at 37&deg;C, 5% CO2.</li>
	<li>The next day transfect Phoenix cells using 12 &mu;g of a vector encoding either Oct3/4, Sox2, Klf4, c-myc, or GFP gene (Addgene plasmids 17217, 17218, 17219, 17220) and 35 &mu;l Fugene 6. Prepare transfection mix in 500 &mu;l of DMEM medium (DMEM high glucose, 2 mM L-glutamine, no FBS and antibiotics). Incubate 20 min. Gently pipette DNA complexes into the 10 cm plates containing 70-80% confluent Phoenix cells.</li>
	<li>Replace media 6 - 8 h post-transfection with DMEM medium (DMEM high glucose, 10% FBS heat inactivated, 2 mM L-glutamine) containing penicillin and streptomycin.</li>
	<li>Subsequently, collect virus-containing media 4 times in 12 h intervals, combine all portions and filtered using a 0.45 &mu;m filter to remove detached cells and debris (media containing viral particles can be kept in the refrigerator for 2 weeks without losing infectious activity, however freezing is not recommended).</li>
	<li>For retroviral transduction, plate human adult fibroblasts (passage 10, Coriell Laboratories) from a frozen stock 24 h prior to the last day of retroviral media collection. Seed the cells on 6-well gelatin covered plates at the density 1x105 cells per well in DMEM high glucose, 10% FBS, 2 mM L-glutamine, penicillin, streptomycin and non-essential amino acids.</li>
	<li>The next day substitute DMEM media with media containing viral particles obtained from Phoenix cells. Add viral media (1 ml of each Oct4, Sox2, Klf4, and c-myc media, 4 ml total) supplemented with 6 &mu;g/ml of polybrene into each well of the 6-well plate of the fibroblast cultures and centrifuge at 1600g for 1h at 20&deg;C. Approximately 12 h after the transduction, replace the viral media with fibroblast culture medium. Perform viral infection and centrifugation 3 times in 24 h intervals.</li>
	<li>Culture fibroblasts in DMEM media for 48 h after last infection. Check the efficiency of the infection by parallel transductions with a retroviral media expressing GFP. Figure 1 illustrates the efficiency of centrifugation-facilitated retroviral infection of human fibroblast.</li>
</ol>
2. Reprogramming
<ol>
	<li>Prepare 6 well plates with &gamma;-irradiated MEF feeder layers by seeding MEF cells at a density of ~2x105 cells per well of the gelatin treated 6 well plate in fibroblast growth medium. The following day split infected human fibroblasts using 0.05% trypsin/EDTA and seed them at the density of ~1.2x104 per well in the fibroblasts growth medium.</li>
	<li>The next day replace media with hES media (DMEM/F12, 20% Knockout Serum Replacement, non-essential amino acids, penicillin/streptomycin, 2 mM L-glutamine, 0.1 mM &beta;-mercaptoethanol, 20 ng/ml basal Fibroblast Growth Factor (bFGF), supplemented with 0.5 mM sodium butyrate for the initial 7 days of the reprogramming. Change media daily.</li>
	<li>Monitor morphological changes in transduced cells daily. Colonies of hiPS-like cells will start to emerge approximately 5 - 10 days after transferring the transduced fibroblasts onto feeder cells. The hiPSC colonies are ready to be isolated approximately 14 - 28 days after plating them on the MEF feeder cells.</li>
</ol>
3. Isolation of hiPSC colonies
<ol>
	<li>A day before picking the hiPSC colonies prepare 24-well plates with &gamma;-irradiated MEF feeder cells (~4x104 cells per well) in fibroblasts growth medium. At this stage hiPSC colonies can also be transferred to feeder free culture using Matrigel and mTeSR1 medium.</li>
	<li>Prior to isolation of hiPSC colonies prepare MEF plates by removing fibroblasts growth medium and rinsing MEFs with PBS to remove traces of FBS. Subsequently add 0.5 ml hES medium (or mTeSR1 medium in the case of Matrigel coated wells) with 10 &mu;M ROCK inhibitor Y27632 to each well 7,8.</li>
	<li>If necessary, remove the fibroblasts surrounding the hiPSC colonies with a 21 gauge needle under a microscope mounted in a laminar flow hood (Figure 2A - C). Rinse the plates with PBS and add fresh hES media containing the Tra-1-81 StainAlive specific antibody (1:200, Stemgent). After 30 min., replace media containing the antibody with fresh hES media supplemented with 10 &mu;M ROCK inhibitor. Examine the plates under the fluorescent microscope and mark Tra-1-81 positive colonies using an objective marker (Figure 2D).</li>
	<li>Cut the Tra-1-81 positive hiPSC colonies under a microscope in a laminar hood, into several small pieces using a 21 gauge needle as shown in Figure 2E and F.</li>
	<li>Using an automatic pipette (P200) transfer fragments of hiPSC colonies into individual wells of the 24-well plate with MEFs or Matrigel. Avoid transferring non-hiPS cells. Place the plates in a 37&deg;C, 5% CO2 incubator and allow the hiPSC colonies to attach for 24-36 h.</li>
	<li>Change hES or mTeSR1 (for colonies grown on Matrigel) media daily. The hiPSC colonies with correct hES-like morphology will be visible 48 h after the initial transfer onto a 24-well plate.</li>
	<li>Manually passage hiPSC colonies every 6 - 8 days onto a 12-well and subsequently on a 6-well plate.</li>
	<li>After clonal expansion and establishing the hiPSC lines, analyze expression of pluripotency markers TRA-1-60, SSEA-4, Oct3/4, Sox2 and Nanog using immunocytochemistry as shown in Figure 3. Evaluate genomic integrity and differentiation potential of the obtained lines using karyotype and teratoma formation analyses 6,9.</li>
</ol>
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.
 
<img alt="Figure 1" src="/files/ftp_upload/3416/3416fig1.jpg" /> 
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.
<img alt="Figure 2" src="/files/ftp_upload/3416/3416fig2.jpg" /> 
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.
<img alt="Figure 3" src="/files/ftp_upload/3416/3416fig3.jpg" /> 
Figure 3. Expression of the pluripotency specific markers Oct3/4, Nanog, Sox2, SSEA4 and Tra-1-60 in hiPSCs was determined by immunocytochemistry.

<ol>
	<li>Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Mali, P., Chou, B. K., Yen, J., Ye, Z., Zou, J., Dowey, S., Brodsky, R. A., Ohm, J. E., Yu, W., Baylin, S. B., Yusa, K., Bradley, A., Meyers, D. J., Mukherjee, C., Cole, P. A., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Butyrate+greatly+enhances+derivation+of+human+induced+pluripotent+stem+cells+by+promoting+epigenetic+remodeling+and+the+expression+of+pluripotency-associated+genes.">Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes.</a> Stem Cells. 28, 713-720 (2011).</li><li>Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B. E., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li><li>Lowry, W. E., Richter, L., Yachechko, R., Pyle, A. D., Tchieu, J., Sridharan, R., Clark, A. T., Plath, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+from+dermal+fibroblasts.">Generation of human induced pluripotent stem cells from dermal fibroblasts.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2883-2888 (2008).</li><li>Dick, E., Matsa, E., Bispham, J., Reza, M., Guglieri, M., Staniforth, A., Watson, S., Kumari, R., Lochmuller, H., Young, L., Darling, D., Denning, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+new+protocols+to+enhance+the+production+and+isolation+of+human+induced+pluripotent+stem+cell+lines.">Two new protocols to enhance the production and isolation of human induced pluripotent stem cell lines.</a> Stem Cell Res. 6, 158-167 (2011).</li><li>Ku, S., Soragni, E., Campau, E., Thomas, E. A., Altun, G., Laurent, L. C., Loring, J. F., Napierala, M., Gottesfeld, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friedreich's+ataxia+induced+pluripotent+stem+cells+model+intergenerational+GAATTC+triplet+repeat+instability.">Friedreich's ataxia induced pluripotent stem cells model intergenerational GAATTC triplet repeat instability.</a> Cell Stem Cell. 7, 631-637 (2010).</li><li>Emre, N., Vidal, J. G., Elia, J., O'Connor, E. D., Paramban, R. I., Hefferan, M. P., Navarro, R., Goldberg, D. S., Varki, N. M., Marsala, M., Carson, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ROCK+inhibitor+Y-27632+improves+recovery+of+human+embryonic+stem+cells+after+fluorescence-activated+cell+sorting+with+multiple+cell+surface+markers.">The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers.</a> PLoS One. 5, e12148-e12148 (2011).</li><li>Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Maherali, N., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3, 595-605 (2008).</li><li>Yu, J., Thomson, J. A., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+Pluripotent+Stem+Cell+Derivation.">Induced Pluripotent Stem Cell Derivation.</a> Essentials of Stem Cell Biology. , 331-337 (2009).</li><li>Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, I. I., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells+free+of+vector+and+transgene+sequences.">Human induced pluripotent stem cells free of vector and transgene sequences.</a> Science. 324, 797-801 (2009).</li><li>Woltjen, K., Michael, I. P., Mohseni, P., Desai, R., Mileikovsky, M., Hamalainen, R., Cowling, R., Wang, W., Liu, P., Gertsenstein, M., Kaji, K., Sung, H. K., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac+transposition+reprograms+fibroblasts+to+induced+pluripotent+stem+cells.">piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.</a> Nature. 458, 766-770 (2009).</li><li>Zhou, W., Freed, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+gene+delivery+can+reprogram+human+fibroblasts+to+induced+pluripotent+stem+cells.">Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells.</a> Stem Cells. 27, 2667-2674 (2009).</li><li>Warren, L., Manos, P. D., Ahfeldt, T., Loh, Y. H., Li, H., Lau, F., Ebina, W., Mandal, P. K., Smith, Z. D., Meissner, A., Daley, G. Q., Brack, A. S., Collins, J. J., Cowan, C., Schlaeger, T. M., Rossi, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+reprogramming+to+pluripotency+and+directed+differentiation+of+human+cells+with+synthetic+modified+mRNA.">Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.</a> Cell Stem Cell. 7, 618-630 (2010).</li><li>Kim, D., Kim, C. H., Moon, J. I., Chung, Y. G., Chang, M. Y., Han, B. S., Ko, S., Yang, E., Cha, K. Y., Lanza, R., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+by+direct+delivery+of+reprogramming+proteins.">Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.</a> Cell Stem Cell. 4, 472-476 (2009).</li><li>Han, S. S., Williams, L. A., Eggan, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructing+and+deconstructing+stem+cell+models+of+neurological+disease.">Constructing and deconstructing stem cell models of neurological disease.</a> Neuron. 70, 626-644 (2011).</li></ol>

The authors have nothing to disclose.

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 re...

<table border="1">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Catalog number </td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>11965</td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>Invitrogen</td>
 <td>11330</td>
 </tr>
 <tr>
 <td>KSR</td>
 <td>Invitrogen</td>
 <td>10828</td>
 </tr>
 <tr>
 <td>Non-essential aminoacids</td>
 <td>Invitrogen</td>
 <td>11140</td>
 </tr>
 <tr>
 <td>Sodium butyrate</td>
 <td>Sigma</td>
 <td>B5887</td>
 </tr>
 <tr>
 <td>Y27632</td>
 <td>Stemgent</td>
 <td>04-0012</td>
 </tr>
 <tr>
 <td>bFGF</td>
 <td>Stemgent</td>
 <td>03-0002</td>
 </tr>
 <tr>
 <td>Tra-1-81 antibody</td>
 <td>Stemgent</td>
 <td>09-0069</td>
 </tr>
 <tr>
 <td>Oct3/4 antibody</td>
 <td>Santa Cruz</td>
 <td>sc-8628</td>
 </tr>
 <tr>
 <td>Nanog antibody</td>
 <td>Cell Signaling Technology</td>
 <td>4903S</td>
 </tr>
 <tr>
 <td>Tra-1-60 antibody</td>
 <td>Millipore</td>
 <td>MAB4360</td>
 </tr>
 <tr>
 <td>Sox2 antibody</td>
 <td>Cell Signaling Technology</td>
 <td>3579S</td>
 </tr>
 <tr>
 <td>SSEA4</td>
 <td>Millipore</td>
 <td>MAB4304</td>
 </tr>
 <tr>
 <td>CF1 MEFs</td>
 <td>Globalstem</td>
 <td>GSC-6201G</td>
 </tr>
 <tr>
 <td>Objective marker</td>
 <td>Nikon</td>
 <td>MBW10010</td>
 </tr>
 <tr>
 <td>Matrigel </td>
 <td>BD Biosciences</td>
 <td>354277</td>
 </tr>
 <tr>
 <td>mTeSR1</td>
 <td>StemCell Technologies</td>
 <td>05850</td>
 </tr>
 <tr>
 <td>&beta;-mercaptoethanol</td>
 <td>Sigma</td>
 <td>M7522</td>
 </tr>
 <tr>
 <td>Fugene 6</td>
 <td>Roche</td>
 <td>11814443001</td>
 </tr>
 <tr>
 <td>polybrene</td>
 <td>Sigma</td>
 <td>H9268</td>
 </tr>
 <tr>
 <td>Object marker</td>
 <td>Nikon</td>
 <td>MBW10010</td>
 </tr>
 </tbody>
</table>

selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

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 (&gt;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.

Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

selecting-isolating-colonies-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

19.8K 次观看.  University of Texas M.D. Anderson Cancer Center. 我们提出了一个高效的人体细胞重编程为人类诱导多能干细胞（hiPSC）用OCT3 / 4，SOX2，KLF4和c-myc（OSKM）的逆转录病毒载体编码和识别正确地重新编程，现场染色hiPSC协议茶1-81抗体。

视频: Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Using Skin Biopsies to Generate Patient-Derived Podocytes

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

Author Spotlight: Generation of Patient-Derived Podocytes from Skin Biopsies  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.