Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.
In 2006, the group of Shinya Yamanaka1 demonstrated for the first time that somatic cells from adult mice and humans can be converted into a pluripotent state by ectopic expression of four reprogramming factors (Oct-4, Sox-2, Klf-4, and c-Myc), generating the so-called Induced Pluripotent Stem Cells (iPSCs)2. Patient-specific iPSCs closely resemble human embryonic stem cells (hESCs) in terms of morphology, proliferation and ability to differentiate into the three-germ cell types (mesoderm, endoderm and ectoderm) while lacking the ethical concerns associated to the use of hESCs and bypassing possible immune rejection3. Thus, iPSCs appear as one of the most important sources of patient-specific cells for basic research, drug screening, disease modeling, evaluation of toxicity, and regenerative medicine purposes4.
Several approaches have been used for iPSC generation: viral integrating vectors (retrovirus5, lentivirus6), viral non-integrating vectors (adenovirus7), Sendai-virus8, BAC transposons9, episomal vectors10, proteins11 or RNA delivery12. Although the use of virus-mediated methods can lead to high efficiency reprogramming, viral vectors integrate into the genome of host cells and therefore potential random insertional mutagenesis, permanent alteration of gene expression, and reactivation of silenced transgenes during differentiation cannot be excluded13.
To make iPSCs safer for regenerative medicine, efforts have been made to derive iPSCs without the integration of exogenous DNA into cellular genomes. Although excisable viral vectors and transposons have been developed, it is still unclear whether short vector sequences, which inevitably remain in the transduced cells after excision, and transposase expression, could induce alteration in cellular function13. Despite its high reprogramming efficiency, Sendai virus represents an expensive approach and reach-through licensing concerns with the company that developed this system have the potential to limit its application in translational studies. Furthermore, the need for direct introduction of proteins and RNA requires multiple delivery of reprogramming molecules with the inherent technical limitations this introduces, and overall reprogramming efficiency is very low14. Of note, cost-effective viral-free and non-integrating methods based on the use of episomal plasmids have been successfully reported for the reprogramming of skin fibroblasts15. Specifically, in the present work we decided to use commercial available integration-free episomal plasmids, as previously reported10,15.
To date, skin fibroblasts represent the most popular donor cell type5. However, other cell sources have been successfully reprogrammed into iPSCs including keratinocytes16, bone marrow mesenchymal stem cells17, adipose stromal cells18, hair follicles19, and dental pulp cells20. The isolation of these cells requires surgical procedures, and several weeks are needed for in vitro cell expansion in order to establish a primary cell culture.
In this light, the selection of starting cell type is critical and it is equally important to be able to produce iPSCs from easily accessible and less invasive tissues such as blood. Both cord blood mononuclear cells (CBMNCs)21,22 and peripheral blood mononuclear cells (PBMNCs)14,22-24 represent suitable sources of cells for the derivation of iPSCs.
Although the efficiency of adult PBMNC reprogramming is 20–50 times lower than that of CBMNCs22, they remain the most convenient cell type for sampling purpose. In fact, PBMNC sampling has the advantage of being minimally invasive, and in addition, these cells do not require extensive expansion in vitro before reprogramming experiments. To date, different protocols have reported that PBMNCs after density gradient separation can be frozen and thawed days to several months after freezing and expanded for few days before reprogramming into iPSCs22,23. Nevertheless, as far as we are aware no reports have described reprogramming of PBMNCs from frozen buffy coats. Importantly, frozen buffy coats collected without density gradient separation represent the most common blood samples stored in large scale biobanks from population studies, thus representing an easily accessible pool of material for iPSC production that avoids further sample collection.
Herein we report for the first time the generation of viral-free iPSCs from human frozen buffy coats, based on a previously described protocol22. In addition, iPSCs were generated from frozen PBMNCs obtained after density gradient separation, as a control protocol for the non-density gradient purified PBMNC results.
Peripheral blood mononuclear cells (PBMNCs) were isolated from human peripheral blood samples of healthy donors after signed informed consent and approval of the Ethical Committee of the Province of South Tyrol. Experiments were conducted in accordance with the principles expressed in the Declaration of Helsinki. All data were collected and analyzed anonymously.
1. Isolation of Peripheral Blood Mononuclear Cells (PBMNCs)
2. Thawing and Plating of PBMNCs (DAY 0)
3. Culture and Expansion of PBMNCs (DAY 2-13)
4. Transfection of PBMNCs with Episomal Plasmids (DAY 14)
Note: Perform the isolation of the four commercial intergation-free episomal plasmids, carrying the pluripotency genes using a commercial kit for plasmid purification and assess the quality of the purified plasmids using an agarose gel analysis, according to the manufacturer’s instructions.
5. Prepare Dishes with Feeder-cells for the Co-culture (DAY 16)
6. Plating Transfected PBMNCs onto MEFs (DAY 17-19)
7. Picking and Expansion of Induced Pluripotent Stem Cells (iPSCs) Clones (DAY 30-35)
Note: At about 30-35 days after transfection, iPSC colonies should be ready for the picking and replating. The reprogramming efficiency should be estimated as the percentage of number of iPSC colonies/total number of electroporated cells, as previously reported26.
8. Characterization of iPSCs (Between 5-15 Passages)
In this study a simple and effective protocol for the generation of viral-free iPSCs by reprogramming of PBMNCs isolated from frozen buffy coats after whole blood centrifugation and comparison with reprogramming of PBMNCs obtained after density gradient separation is reported. Figure 1A shows a schematic representation of the detailed protocol. After thawing, the isolated PBMNCs, showing a typical rounded shape, are expanded in specific blood culture medium for 14 days (Figure 1B) and then transfected with episomal plasmids. After 10-15 days post-transfection, small rounded bright colonies with defined margins and embryonic stem cell-like morphology start to appear (Figure 1C). Approximately 20-30 days after transfection, iPSC colonies become highly compact, with sharp edges, increase their size and are ready for picking and expansion (Figure 1D). After the first passaging steps (2-3 passages), iPSC colonies maintain their undifferentiated state and show a typical human embryonic-stem cell morphology. During the first passages, manual picking is a more efficient expansion way compared to enzyme dissociation in order to select fully undifferentiated, compact and tightly packed colonies (Figures 2A). Larger magnification of iPSC colonies shows individual cells within the colonies and the high nucleus to cytoplasm ratio is more easily observed (Figure 2B). After initial mechanical picking, selected iPSC clones are expanded with enzyme dissociation using Collagenase IV. After 10-15 passages of in vitro expansion, cytogenetic analysis is conducted on iPSC colonies. About 20 metaphases from at least two independent cultures, at approximately 300–400 band level, are analyzed and all samples show a normal karyogram (Figure 2C). This observation suggests that iPSCs are genetically stable.
After in vitro expansion (5-10 passages), iPSCs are characterized for the expression of stemness markers and their pluripotency potential. Figures 3A, B show that iPSCs are positive for alkaline phosphatase staining. Using qRT-PCR analysis, we have verified that iPSCs express endogenous pluripotent genes (SOX-2, OCT3/4, NANOG) at significantly higher levels than starting PBMNCs, while expression levels of endogenous C-MYC and KLF-4 are similar before and after iPSC reprogramming (Figure 3C). After only 5 passages in culture, expression of exogenous episomal transgenes cannot be detected in iPSCs, thus indicating the successful activation of endogenous pluripotent genes. PBMNCs 5 days after transfection, with strong transgene expression level, evaluated by episomal specific primers for EBNA-1, are used as positive control. PBMNCs that are never exposed to plasmids carrying the 4 exogenous pluripotency genes, are used as negative control(Figure 3D). Finally, immunofluorescence analysis reveals that iPSCs express pluripotency markers, such as OCT3/4, SOX-2, SSEA-4, TRA-1-60, and TRA-1-81 (Figure 4).
iPSCs are differentiated into embryoid bodies (EBs) using a spontaneous differentiation protocol, as shown in Figure 5A, in order to evaluate their in vitro pluripotency potential. qRT-PCR experiments reveal that iPSCs are able to differentiate into cells of the three germ layers; indeed, EBs obtained after 20 days of differentiation display significant up-regulation of lineage markers representative of the endoderm (SOX-7, AFP), mesoderm (CD31, ACTA-2, CDH-5, RUNX-1) and ectoderm (KTR-14, TH, GABRR-2) (Figure 5B). Confocal microscopy analysis shows that single-cell dissociated beating clusters express typical cardiomyocyte markers such as Troponin I (Tn-I) and α-Actinin, organized in a clear sarcomeric pattern (Figure 6A). In addition, immunofluorescence analysis of EBs shows a positive staining for the neuron-specific marker class III β-Tubulin (TUJ1) as well for the dopaminergic neuronal marker tyrosine hydroxylase (TH) (Figure 6B).
Figure 1. Protocol for the generation of iPSCs from human peripheral blood and cell morphological changes during the protocol. (A) Diagram of the protocol used to generate iPSCs obtained from frozen PBMNCs after DGS and PBMNCs isolated from frozen buffy coats, using a single transfection of non-integrating episomal plasmids. (B) Representative images of proliferating PBMNCs obtained from DGS and buffy coats after thawing and expansion in culture for 14 days, before being transfected. Scale bar 100 µm. (C) Examples of iPSC colonies one week after the cells are plated on MEFs. Scale bar 500 µm. (D) Representative images of iPSC colonies at 30-35 days after re-plating on a MEF feeder-layer. Scale bar 500 µm. iPSCs = induced pluripotent stem cells; DGS = density gradient separation; PBMNCs = peripheral blood mononuclear cells; MEFs = mouse embryonic fibroblasts; GFs = growth factors; bFGF = basic fibroblast growth factor; NaB = sodium butyrate. Please click here to view a larger version of this figure.
Figure 2. Morphology and karyotype analysis. (A) Representative images of iPSC colonies after 2-3 manual passaging steps. Scale bar 500 µm. (B) Larger magnification of iPSC colonies. Scale bar 200 µm. (C) Representative normal karyograms from iPSCs after 10-15 passages of in vitro expansion. Please click here to view a larger version of this figure.
Figure 3. iPSC characterization: alkaline phosphatase activity, re-expression of endogenous pluripotency genes of iPSCs and evaluation of transgene silencing. (A) and (B) Representative images showing the positive staining for alkaline phosphatase. Scale bar 500 µm. (C) The bar graph showing the re-expression of endogenous pluripotency genes (C-MYC, KLF-4, SOX-2, OCT3/4 and NANOG) in both iPSCs obtained from DGS and buffy coats, using qRT-PCR analysis (n=4, Student t-test : * p <0.05, § p <0.005 vs corresponding PBMNCs). (D) qRT-PCR with episomal-specific primers (EBNA-1) (relative to control FBOX15 primers) shows no genome-integration of episomal vectors. (n = 4, Student t-test: * p <0.001 vs corresponding PBMNCs 5-day transfection). Please click here to view a larger version of this figure.
Figure 4. Immunofluorescence analysis of embryonic stem cells pluripotency markers. Representative immunofluorescence staining showing significant expression of pluripotency proteins SSEA-4, TRA-1-60, TRA-1-81, OCT3/4 and SOX-2. Nuclei are counterstained with DAPI. Scale bar 200 µm for iPSC DGS (A) and iPSC Buffy coat (B). Scale bar 100 µm for iPSC Buffy coat (A), iPSC DGS (B) and (C). Please click here to view a larger version of this figure.
Figure 5. Protocol for the differentiation of iPSCs and expression of three germ layer genes in EBs obtained from iPSCs. (A) Schematic representation of the protocol inducing spontaneous differentiation of iPSCs from PBMNCs after DGS and buffy coats in EBs. (B) qRT-PCR experiments showing the expression of all three germ layer genes: endoderm (SOX-7, AFP), mesoderm (CD31, ACTA-2, CDH-5, RUNX-1), and ectoderm (KRT-14, TH, GABRR-2) in EBs after 20 days of differentiation (n = 4, Student t-test: * p <0.05 vs corresponding iPSCs counterpart). EBs = embryoid bodies. Please click here to view a larger version of this figure.
Figure 6. Confocal microscopy analysis of cardiac and neuronal proteins in EBs at day 20 of differentiation obtained from PBMNCs after DGS and buffy coats-derived iPSCs. (A) Representative images (maximum projection of confocal image stack) for cardiac sarcomeric proteins α-Actinin and Troponin I (Tn-I) in single-cell dissociated beating areas (scale bar 10 µm) and (B) for neuron-specific marker class III β-Tubulin (TUJ1) and the dopaminergic neuronal marker tyrosine hydroxylase (TH) (scale bar 20 µm). Nuclei are counterstained with DAPI. Please click here to view a larger version of this figure.
Primer | Sequence |
C-MYC fw | 5’-AGAAATGTCCTGAGCAATCACC-3’ |
C-MYC rev | 5’-AAGGTTGTGAGGTTGCATTTGA-3’ |
KLF-4 fw | 5′-ATAGCCTAAATGATGGTGCTTGG-3’ |
KLF-4 rev | 5′- AACTTTGGCTTCCTTGTTTGG-3’ |
SOX-2 fw | 5′- GGGAAATGGGAGGGGTGCAAAAGAGG-3’ |
SOX-2 rev | 5′- TTGCGTGAGTGTGGATGGGATTGGTG-3 |
OCT3/4 fw | 5′- GACAGGGGGAGGGGAGGAGCTAGG-3’ |
OCT3/4 rev | 5′- CTTCCCTCCAACCAGTTGCCCCAAAC-3’ |
NANOG fw | 5′-TGCAAGAACTCTCCAACATCCT-3’ |
NANOG rev | 5′-ATTGCTATTCTTCGGCCAGTT-3’ |
SOX-7 fw | 5′-TGAACGCCTTCATGGTTTG-3’ |
SOX-7 rev | 5′-AGCGCCTTCCACGACTTT-3’ |
AFP fw | 5′-GTGCCAAGCTCAGGGTGTAG -3’ |
AFP rev | 5′-CAGCCTCAAGTTGTTCCTCTG-3’ |
CD31 fw | 5′-ATGCCGTGGAAAGCAGATAC-3’ |
CD31 rev | 5′-CTGTTCTTCTCGGAACATGGA-3’ |
ACTA-2 fw | 5′-GTGATCACCATCGGAAATGAA-3’ |
ACTA-2 rev | 5′-TCATGATGCTGTTGTAGGTGGT-3’ |
CDH-5 fw | 5′-GAGCATCCAGGCAGTGGTAG-3’ |
CDH-5 rev | 5′-CAGGAAGATGAGCAGGGTGA-3’ |
RUNX-1 fw | CCCTAGGGGATGTTCCAGAT-3' |
RUNX-1 rev | TGAAGCTTTTCCCTCTTCCA-3' |
KRT-14 fw | 5′-CACCTCTCCTCCTCCCAGTT-3’ |
KRT-14 rev | 5′-ATGACCTTGGTGCGGATTT-3’ |
TH fw | 5′-TGTACTGGTTCACGGTGGAGT-3’ |
TH rev | 5′-TCTCAGGCTCCTCAGACAGG-3’ |
GABBR-2 fw | 5′-CTGTGCCTGCCAGAGTTTCA-3’ |
GABBR-2 rev | 5′-ACGGCCTTGACGTAGGAGA-3’ |
EBNA-1 fw | 5′-ATCAGGGCCAAGACATAGAGATG-3’ |
EBNA-1 rev | 5′-GCCAATGCAACTTGGACGTT-3’ |
FBX-15 fw | 5′-GCCAGGAGGTCTTCGCTGTA-3’ |
FBX-15 fw | 5′-AATGCACGGCTAGGGTCAAA-3’ |
GAPDH fw | 5′-CCACCCATGGCAAATTCC-3’ |
GAPDH rev | 5’-TCGCTCCTGGAAGATGGTG-3’ |
Table 1: List of qRT-PCR primers. Primer sequences used for evaluation of endogenous pluripotency gene expression, transgene silencing and expression of three germ layer markers.
In the past, the only way to obtain human pluripotent stem cells carrying a particular genetic mutation was to recruit parents undergoing pre-implantation genetic diagnosis and generate embryonic stem cells from their discarded blastocysts31,32. Using a reprogramming approach, researchers can now generate iPSCs from patients carrying virtually any genotype. The possibility to start from a patient-specific cell line and an easily accessible source is very important since it allows an investigation of the pathophysiology of disorders and subsequent identification of novel therapeutic approaches.
In the present study, we combined a plasmid approach15 with a method already applied to produce iPSCs from PBMNCs previously frozen after density gradient separation22 and successfully generated virus-free iPSCs from frozen buffy coat PBMNCs. The use of buffy coats instead of density gradient separated PBMNCs allows us to reduce costs, working time and manual steps for operators during sample processing. The set-up of a low-cost protocol using PBMNCs from frozen buffy coats is essential for studies with elevated number of participants and for sample collections in multi-center studies. Importantly, the ability to produce iPSCs from frozen buffy coats allows access to numerous frozen biosamples already stored in blood banks, using a simple, cost-effective and not so time consuming method for sample collection.
This induction method may be useful for the derivation of patient-specific integration-free iPSCs, where colonies are characterized by the absence of episomal transgene after only few passages (≥5 passages), in both starting materials. Remarkably, we observed that all the selected iPSC colonies obtained from both types of starting material display comparable pluripotency features and the preservation of cellular and molecular similarities to hESCs, indicating a successful iPSC derivation and a consistent reproducibility of the protocol.
Moreover, we have successfully generated iPSCs from more than 10 human skin fibroblast and 3 cardiac mesenchymal stromal cell lines (both from healthy donors and patients with cardiac and skeletal muscle diseases) using this method (data not shown), thus suggesting that the described protocol can be used for the successful reprogramming of different cell types. However, the choice of blood as starting material is advantageous compared to fibroblasts, especially when considering that extensive expansion of fibroblasts in vitro may affect the efficiency of reprogramming, based on fibroblast passage number and proliferation rate33,34. Our protocol also allows the generation of transgene-free iPSCs from frozen buffy coats after a minimal culture time, thus reducing by about 3-4 weeks the time needed for derivation of iPSCs compared to other sources, such as fibroblasts. Furthermore, recent studies revealed that blood cell-derived iPSCs display an epigenetic signature that more closely resembles hESCs than those obtained from mesenchymal stem cells (MSC)/fibroblasts14,35.
Despite of the successful generation of iPSCs from frozen buffy coats, a few limitations need to be considered in this protocol. Before reprogramming induction, the amplification of PBMNCs from frozen buffy coats could represent a critical step of the protocol. The quality of starting material can strongly influence the selection and isolation of PBMNCs, in order to achieve sufficient cell numbers for reprogramming procedure (at least 2 x 106 cells for electroporation). This is mostly due to intrinsic biological variability of the samples, but also to technical issues. Indeed, the amplification of PBMNCs from frozen buffy coats is less efficient compared to PBMNCs obtained from density gradient separation, due to a strong dependence on manual operator variability. In order to enhance reproducibility and efficiency of PBMNC amplification from frozen buffy coats without density gradient separation, the use of automated systems is strongly recommended to collect buffy coat fractions. Although it is more difficult to expand a sufficient number of PBMNCs from buffy coats than PBMNCs after density gradient separation, the efficiency of reprogramming (about 0.001-0.0005%) of the two starting materials is comparable, according to blood reprogramming efficiency previously reported14,23. Since the reprogramming efficiency is low for regenerative medicine purposes, the use of improved EBNA1/OriP (from Epstein-Barr virus, EBV) episomal vectors may increase the number of iPSC colonies for future cell therapy development22,36. Additionally, a MEF feeder layer for iPSC generation and maintenance is used in our protocol. In order to obtain clinical-grade iPSCs, a feeder-free culture could be an alternative method for further studies.
In conclusion, this protocol represents a valid method to generate iPSCs from an easily accessible patient cell source with the great advantage of using a non-integrative system that can potentially be used for the derivation of clinical-grade iPSCs, not only for disease modelling but also for a future personalized medicine.
The authors have nothing to disclose.
The study was supported by the Ministry of Health and Department of Educational Assistance, University and Research of the Autonomous Province of Bolzano and the South Tyrolean Sparkasse Foundation.
Sodium Citrate buffered Venosafe Plastic Tube | Terumo | VF-054SBCS07 | |
Ammodium chloride | Sigma-Aldrich | A9434 | |
Potassium bicarbonate | Sigma-Aldrich | 60339 | |
EDTA disodium powder | Sigma-Aldrich | E5134 | 0.5 M solution |
Ficoll-Paque Premium | GE Healthcare Life Sciences | 17-5442-02 | Polysucrose solution for density gradient centrifugation |
Iscove's modified Dulbecco's medium (IMDM) | Gibco | 21056-023 | No phenol red |
Ham's F-12 | Mediatech | 10-080-CV | |
Insulin-Transferrin-Selenium-Ethanolamine (ITS -X) | Gibco | 51500-056 | 1X (Stock: 100X) |
Chemically Defined Lipid Concentrate | Gibco | 11905031 | 1X (Stock: 100X) |
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A9418 | |
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate | Sigma-Aldrich | A8960 | |
1-Thioglycerol | Sigma-Aldrich | M6145 | Final Concentration at 200 µM |
Recombinant Human Stem Cell Factor (SCF) | PeproTech | 300-07 | 100 ng/ml (Stock:100 µg/ml) |
Recombinant Human Interleukin-3 (IL-3) | PeproTech | 200-03 | 10 ng/ml (Stock: 10 µg/ml) |
Recombinant Human Insulin-like Growth Factor (IGF-1) | PeproTech | 100-11 | 40 ng/ml (Stock: 40 µg/ml) |
Recombinant Human Erythropoietin (EPO) | R&D Systems | 287-TC-500 | 2 U/ml (Stock: 50 U/ml) |
Dexamethasone | Sigma-Aldrich | D2915 | 1 μM (Stock: 1 mM) |
Human Holo-Transferrin | R&D Systems | 2914-HT | 100 μg/ml (Stock: 20 mg/ml) |
Amniomax II | Gibco | 11269016 | Medium for cytogenetic analysis |
mTeSR1 | StemCell Technologies | 5850 | Medium for iPSC feeder-free culture |
Knockout DMEM | Gibco | 10829-018 | |
Knockout Serum Replacement | Gibco | 10828-028 | |
Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140-122 | |
L-Glutamine (200 mM) | Gibco | 25030-024 | |
MEM Non-Essential Amino Acids Solution (100X) | Gibco | 11140-050 | |
2-Mercaptoethanol | Gibco | 31350-010 | 0.1 mM (Stock: 50 mM) |
Sodium Butyrate | Sigma-Aldrich | B5887 | 0.25 mM (Stock: 0.5 M) |
Recombinant Human FGF basic, 145 aa | R&D Systems | 4114-TC | 10 ng/ml (Stock: 10 µg/ml) |
Y-27632 dihydrochloride | Sigma-Aldrich | Y0503 | 10 µM (Stock: 10 mM) |
Fetal Defined Bovine Serum | Hyclone | SH 30070.03 | |
EmbryoMax 0.1% Gelatin Solution | Merck-Millipore | ES-006-B | |
Matrigel Basement Membrane Matrix Growth Factor Reduced | BD Biosciences | 354230 | |
Collagenase, Type IV | Gibco | 17104-019 | 1 mg/ml (Stock: 10 mg/ml) |
Accutase | PAA Laboratories GmbH | L11-007 | Cell detachment solution |
Mouse Embryonic Fibroblast (CF1) | Global Stem | GSC-6201G | 1*106 cells/6 well plate |
Plasmid pCXLE-hOCT3/4-shp53-F | Addgene | 27077 | 1 µg (Stock: 1 µg/µl) |
Plasmid pCXLE-hSK | Addgene | 27078 | 1 µg (Stock: 1 µg/µl) |
Plasmid pCXLE-hUL | Addgene | 27080 | 1 µg (Stock: 1 µg/µl) |
Plasmid pCXLE-EGFP | Addgene | 27082 | 1 µg (Stock: 1 µg/µl) |
Alkaline Phosphatase Staining Kit | Stemgent | 00-0009 | |
Anti-Stage-Specific Embryonic Antigen-4 (SSEA-4) Antibody | Merck-Millipore | MAB4304 | 1/250 |
Anti-TRA-1-60 Antibody | Merck-Millipore | MAB4360 | 1/250 |
Anti-TRA-1-81 Antibody | Merck-Millipore | MAB4381 | 1/250 |
Anti-Oct-3/4 Antibody | Santa Cruz Biotechnology | sc-9081 | 1/500 |
Anti-Nanog Antibody | Santa Cruz Biotechnology | sc-33759 | 1/500 |
Anti-Troponin I Antibody | Santa Cruz Biotechnology | sc-15368 | 1/500 |
Anti-α-Actinin (Sarcomeric) Antibody | Sigma-Aldrich | A7732 | 1/250 |
Neuronal Class III ß-Tubulin (TUJ1) Antibody | Covance Research Products Inc | MMS-435P-100 | 1/500 |
Anti-Tyrosine Hydroxylase (TH) Antibody | Calbiochem | 657012 | 1/200 |
Alexa Fluor 488 Goat Anti-Mouse | Molecular Probes | A-11029 | 1/1000 |
Alexa Fluor 555 Goat Anti-Rabbit | Molecular Probes | A-21429 | 1/1000 |
Ultra-Low Attachment Cell Culture 6-well plate | Corning | 3471 | |
Trizol Reagent | Ambion | 15596-018 | reagent for RNA extraction |
SuperScript VILO cDNA Synthesis Kit | Invitrogen | 11754050 | Reverse transcriptase kit |
iTaq Universal SYBR Green Supermix | Bio-Rad | 172-5124 | |
CFX96 Real-Time PCR Detection System | Bio-Rad | 185-5195 | |
Experion Automated Electrophoresis System | Bio-Rad | 700-7000 | Instrument to check RNA integrity |
Experion RNA Highsense Analysis kit | Bio-Rad | 7007105 | Reagent kit to check RNA integrity |
Dissecting microscope (SteREO Discovery V12 ) | Zeiss | 495007 | |
NeonTransfection System 100 µL Kit | Invitrogen | MPK10025 | Reagent kit for electroporation |
Neon Transfection System | Invitrogen | MPK5000 | Instrument used for electroporation |
NanoDrop UV/Vis Spectrophotometer | Thermo Scientific | ND-2000 | Instrument for DNA/RNA quantification |
EndoFree Plasmid Maxi Kit | Qiagen | 12362 | Plasmid purification kit |