Name: generation and maintenance of primate induced pluripotent stem cells derived from urine
Uploaded: 2023-07-28T12:40:00
Description: Watch this Scientific Journal Video about Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine at JoVE.com

This work was supported by DFG EN 1093/5-1 (project number 458247426). M.O. was supported by JSPS Overseas Research Fellowship. All figures were created with BioRender.com. Flow cytometry was performed with the help of the Core Facility Flow Cytometry at Biomedical Center Munich. We would like to thank Makoto Shida and Tomoyo Muto from ASHBi,&#160;Kyoto University, for support of videography.

This experimental procedure was approved by the responsible ethic committee on human experimentation (20-122, Ethikkommission LMU M&#252;nchen). All experiments were performed in accordance with relevant guidelines and regulations. 
NOTE: Approval must be obtained from the appropriate ethical committee before starting experiments dealing with human and NHP samples. All experimental procedures must be performed in accordance with relevant guidelines and regulations. Each of the following steps should be performed usi...

iPSCs are valuable cell types as they allow the generation of otherwise inaccessible cell types in vitro. As the starting materials for reprogramming, for example, fibroblasts are not easily available from all primate species, this paper presents a protocol for the generation of iPSCs from urine-derived cells. These cells can be obtained in a non-invasive manner, even from non-sterile primate urine samples, by supplementing the culture medium with broad-spectrum antibiotics.
Several c...

Genomic comparisons of human and non-human primates (NHPs) are crucial to understand our evolutionary history and the evolution of human-specific traits1. Additionally, these comparisons allow for the inference of function by identifying conserved DNA sequences2, e.g., to prioritize disease-associated variants3. Comparisons of molecular phenotypes such as gene expression levels are crucial to better interpret genomic comparisons and to discover, for example, cellular phenotypic differences. Furthermore, they have - similar to comparisons at the DNA level - the potential to infer functional relevance, and hence to better interpret medically relevant variation within humans4. The incorporation of comprehensive molecular phenotypic data into these comparative studies requires appropriate biological resources (i.e., orthologous cells across species). However, ethical and practical reasons make it difficult or impossible to access such comparable cells, especially during development. Induced pluripotent stem cells (iPSCs) allow for the generation of such inaccessible cell types in vitro5,6, are experimentally accessible, and have been used for primate comparisons6,7,8,9,10,11,12,13,14.
To generate iPSCs, one needs to acquire the primary cells to be reprogrammed. Cells isolated from urine have the advantage that they can be sampled non-invasively from primates, and that they can be readily reprogrammed, probably due to their stem cell-like molecular profiles15. The culture conditions to maintain primate iPSCs are as important as reprogramming; classically, the culture of human pluripotent stem cells required a non-defined, serum-based medium and co-culture of mouse embryonic fibroblasts - so-called feeder cells - that provide essential nutrients and a scaffold for embryonic stem cells (ESCs)16. Since the development of chemically defined and feeder-free culture systems17,18, there are now various options of commercially available iPSC culture media and matrices. However, most of these culture conditions have been optimized for human ESCs and iPSCs, and hence might work less well in NHP iPSC culture. In this video protocol,&#160;we provide instructions to generate and maintain human and NHP iPSCs derived from urinary cell culture.
Since the first report of iPSC generation&#160;by the forced expression of defined factors in fibroblasts in 2006, this method has been applied to many different cell types of various origins19,20,21,22,23,24,25,26,27,28,29,30,31,32. Among them, only urine-derived cells can be obtained in a completely non-invasive manner. Based on the previously described protocol by Zhou et al.33, one can isolate and expand cells from primate urine even from non-sterile samples, by supplementing broad-spectrum antibiotics15. Notably, urine-derived cells sampled by this protocol exhibit a high potential to produce iPSCs, within a shorter period of time&#160;(colonies become visible in 5-15 days) than the conventional reprogramming of fibroblasts (20-30 days, in our experience), and with a sufficiently high success rate. These urine-derived cells were classified as the mixed population of mesenchymal stem cell-like cells and bladder epithelial cells, causing the high reprogramming efficiency15.
In addition to the variation in primary cells, the reprogramming methods to generate iPSCs also vary&#160;according to the purpose of usage. Conventional reprogramming procedures for human somatic cells were carried out by the overexpression of reprogramming factors with retrovirus or lentivirus vectors, which allowed the integration of exogenous DNA in the genome5,34,35. To keep the generated iPSCs genomically intact, researchers have developed a wide variety of non-integration systems - excisable PiggyBac vector36,37, episomal vector38,39, non-integrating virus vectors such as Sendai virus40 and adenovirus41, mRNA transfection42, protein transfection43,44, and chemical compound treatment45. Due to the efficiency and ease in handling, the Sendai virus-based reprogramming vectors are used in this protocol. Infection of primary cells is performed in a 1 h suspension culture of cells and viruses at a multiplicity of infection (MOI) of 5 prior to plating. This modified step could increase the likelihood of contact between cell surfaces and viruses, compared to the conventional method in which the viruses are added directly to the adherent cell culture, and thus yield more iPSC colonies15.
Passaging of human and NHP pluripotent stem cells can be done by clump passaging and single-cell passaging. Ethylenediaminetetraacetic acid (EDTA) is a cost-efficient chelating agent that binds calcium and magnesium ions, and thus prevents the adherent activity of cadherin and integrin. EDTA is also used as a mild, selective dissociation reagent, as undifferentiated cells detach before differentiated cells due to their different adhesion molecules. Complete dissociation induces massive cell death of primate iPSCs via the Rho/Rho-associated coiled-coil containing protein kinase (Rho/Rock)-mediated myosin hyperactivation. Therefore, supplementing the culture medium with a Rho/Rock inhibitor is essential for experiments that require single cells in suspension46,47. In this protocol, we recommend clump passaging as the routine passaging method&#160;and recommend single-cell passaging only when it is necessary, e.g., when seeding of defined cell numbers is required, or during sub-cloning.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accumax&#8482; cell detachment solution (Detachment solution)</td>
			<td>Sigma-Aldrich</td>
			<td>SCR006</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B-Solution</td>
			<td>Merck</td>
			<td>A2941-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human TRA-1-60 Mouse Antibody&#160;</td>
			<td>Stem Cell Technologies</td>
			<td>60064</td>
			<td>Dilution: 1/200</td>
		</tr>
		<tr>
			<td>Anti-Human TRA-1-60 PE-conjugated Antibody&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-122-965</td>
			<td>Dilution: 1/50</td>
		</tr>
		<tr>
			<td>Bambanker&#8482; (Cell freezing medium)</td>
			<td>Nippon Genetics</td>
			<td>BB01</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A3059-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture multiwell plate, 12-well CELLSTAR</td>
			<td>Greiner BIO-ONE</td>
			<td>665180</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess&#8482; II automated cell counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoKing&#174; 1.5 mL Tubes with 2D Barcode (Cryotubes)</td>
			<td>Sued-Laborbedarf</td>
			<td>52 95-0213</td>
			<td>Different types of Cryotubes can be used for freezing. The 2D barcode tubes have the advantage that the sample info can be stored in a database with unique tube information.</td>
		</tr>
		<tr>
			<td>CytoTune&#8482; EmGFP Sendai Fluorencence Reporter (GFP Sendai virus)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A16519</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoTune&#8482;-iPS 2.0 Sendai Reprogramming Kit (Sendai virus reprogramming kit)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A16518</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI 4&#39;,6-Diamidine-2&#39;-phenylindole dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>10236276001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM High Glucose</td>
			<td>TH.Geyer</td>
			<td>L0102</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 w L-glutamine</td>
			<td>Fisher Scientific</td>
			<td>15373541</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor&#8482; 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21202</td>
			<td>Dilution: 1/500</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor&#8482; 594</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21207</td>
			<td>Dilution: 1/500</td>
		</tr>
		<tr>
			<td>DPBS w/o Calcium w/o Magnesium</td>
			<td>TH.Geyer</td>
			<td>L0615-500</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM Recombinant Polyclonal Rabbit Antibody (22 HCLC)</td>
			<td>Thermo Fisher Scientific</td>
			<td>710524</td>
			<td>Dilution: 1/500</td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid (EDTA)</td>
			<td>Carl Roth</td>
			<td>CN06.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Tube 15 mL conical bottom</td>
			<td>Greiner BIO-ONE</td>
			<td>188271-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Tube 50 mL conical bottom</td>
			<td>Greiner BIO-ONE</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated, Brazil (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo V10.8.2</td>
			<td>FlowJo&#160;</td>
			<td>663441</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex&#8482; LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1413301</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX&#8482; Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell&#8482; 240i CO2 incubator</td>
			<td>Fisher Scientific</td>
			<td>16416639</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraeus HeraSafe safety cabinet</td>
			<td>Kendro</td>
			<td>51017905</td>
			<td></td>
		</tr>
		<tr>
			<td>Human EGF, premium grade</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-749</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ&#160;</td>
			<td>Fiji</td>
			<td>Version 2.9.0</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140035</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifugation tube PP, 1.5 mL</td>
			<td>Nerbe Plus</td>
			<td>04-212-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Nikon eclipse TE2000-S</td>
			<td>Nikon</td>
			<td>TE2000-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-alpha-Fetoprotein antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1368</td>
			<td>Dilution: 1/100</td>
		</tr>
		<tr>
			<td>Mouse anti-alpha-Smooth Muscle Actin antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1420</td>
			<td>Dilution: 1/100</td>
		</tr>
		<tr>
			<td>Mouse anti-beta-III Tubulin antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1195</td>
			<td>Dilution: 1/100</td>
		</tr>
		<tr>
			<td>mTeSR&#8482; 1</td>
			<td>STEMCELL Technolgies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanog (D73G4) XP Rabbit mAb&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>4903S</td>
			<td>Dilution: 1/400</td>
		</tr>
		<tr>
			<td>Normocure&#8482; (Antimicrobial Reagent)</td>
			<td>Invivogen</td>
			<td>ant-noc</td>
			<td></td>
		</tr>
		<tr>
			<td>Oct-4 Rabbit Antibody&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>2750S</td>
			<td>Dilution: 1/400</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>441244-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10.000 U/ml) (PS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Penicillin-Streptomycin mix contains 100 U/mL Penicillin and 100 &#181;g/mL Streptomycin.</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-basic</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human PDGF-AB</td>
			<td>PeproTech</td>
			<td>100-00AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated benchtop centrifuge</td>
			<td>SIGMA&#160;</td>
			<td>4-16KS</td>
			<td></td>
		</tr>
		<tr>
			<td>Renal Epithelial Cell Basal Medium</td>
			<td>ATCC</td>
			<td>PCS-400-030</td>
			<td></td>
		</tr>
		<tr>
			<td>Renal Epithelial Cell Growth Kit</td>
			<td>ATCC</td>
			<td>PCS-400-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Sox2 (L1D6A2) Mouse mAb #4900</td>
			<td>Cell Signaling Technology</td>
			<td>4900S</td>
			<td>Dilution: 1/400</td>
		</tr>
		<tr>
			<td>SSEA4 (MC813) Mouse mAb</td>
			<td>NEB</td>
			<td>4755S</td>
			<td>Dilution: 1/500</td>
		</tr>
		<tr>
			<td>StemFit&#174; Basic02</td>
			<td>Nippon Genetics</td>
			<td>3821.00</td>
			<td>The production of this medium was discontinued, use StemFit Basic04CT for human cell lines or StemFit Basic03 for non-human primates instead.</td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE&#8482; Select Enzyme (1x), no phenol red (Dissociation enzyme)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12563011</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath Precision GP 05</td>
			<td>Thermo Fisher Scientific</td>
			<td>TSGP05</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632, Dihydrochloride Salt (Rock Inhibitor)</td>
			<td>Biozol</td>
			<td>BYT-ORB153635</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody dilution buffer</td>
			<td></td>
			<td></td>
			<td>For composition see the supplementary table S1</td>
		</tr>
		<tr>
			<td>Blocking buffer</td>
			<td></td>
			<td></td>
			<td>For composition see the supplementary table S1</td>
		</tr>
		<tr>
			<td>REMC medium</td>
			<td></td>
			<td></td>
			<td>For composition see the supplementary table S1</td>
		</tr>
		<tr>
			<td>Primary urine medium</td>
			<td></td>
			<td></td>
			<td>For composition see the supplementary table S1</td>
		</tr>
		<tr>
			<td>PSC culture medium</td>
			<td></td>
			<td></td>
			<td>For composition see the supplementary table S1</td>
		</tr>
		<tr>
			<td>PSC generation medium</td>
			<td></td>
			<td></td>
			<td>For composition see the supplementary table S1</td>
		</tr>
		<tr>
			<td>Urine wash buffer</td>
			<td></td>
			<td></td>
			<td>For composition see the supplementary table S1</td>
		</tr>
	</tbody>
</table>

generation and maintenance of primate induced pluripotent stem cells derived from urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

When isolating cells from human and NHP urine, different types of cells can be identified directly after isolation. Squamous cells, as well as various smaller round cells, get excreted with the urine; female urine contains far more squamous cells than male urine (Figure 1B - Day 0; Supplementary Figure S1). After 5 days of culture in primary urine medium, the first adherent proliferating cells can be seen (Figure 1A,B&#160;- Day...

Watch this Scientific Journal Video about Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine at JoVE.com

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular ...

Primate IPSCs are useful but often difficult to obtain due to ethical and technical issues. This protocol gives a most accessible path to the generation and maintenance of primate IPSCs. The use of urinary cells as a primary source of IPSCs has a big advantage as they can be obtained in a completely non-invasive manner without any special techniques.Demonstrating the procedure will be Jessica Radmer, a PhD student from Wolfgang M.S.Laboratory. To begin, centrifuge the urine containing tube at 400G for 10 minutes. Then carefully aspirate the supernatant, leaving approximately one milliliter in the tube and resuspend the pellet in it.Wash the cells by adding 10 milliliters of urine wash buffer containing amphotericin and carefully mix the suspension using a serological pipette. After centrifugation, carefully aspirate the supernatant, leaving approximately less than 0.2 milliliters in the tube. Resuspend the cell pellet in one milliliter of primary urine medium containing amphotericin per 50 milliliters of initially processed urine.Remove gelatin and add one milliliter of the suspension into a well of gelatin coated 12 well plate. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide. To prepare a basement membrane matrix coated 12 well plate, add 500 microliters of basement membrane matrix per well.Move the plate to distribute the liquid and incubate it at 37 degrees Celsius for one hour. After incubation, replace the basement membrane matrix with 900 microliters of REMC medium and store it at 37 degrees Celsius until use. Calculate the volume of Sendai viruses needed for a multiplicity of infection of five using the equation.Quickly thaw the components of the Sendai reprogramming kit in the water bath tempered at 37 degrees Celsius. Add REMC medium for a total of 100 microliters to a tube before adding the calculated volume of Sendai viruses and mixing. After dissociation of the urinary cells as described in the manuscript, count the cells using the cell counter and transfer the desired number of cells to a 1.5 milliliter tube.Obtain the pellet by centrifugation and resuspend it in 100 microliters of the prepared Sendai virus mixture. Incubate the tube for one hour at 37 degrees Celsius for suspension infection. After incubation, add REMC to a total of one milliliter before seeding the cell suspension in the basement membrane matrix coated 12 well plate.After transduction, change the medium every second day until the development of induced pluripotent stem cells, or IPSC, colonies with switching to PSC generation medium on day five. When the IPSC colonies grow large enough for clump passaging, aspirate the medium from the cultured cells and carefully wash the cells with 500 microliters of DPBS. After removing the DPBS, add 500 microliters of 0.5 millimolar EDTA to the well and incubate for two to five minutes.Carefully observe the cells under the microscope until the colonies start detaching. When the edges of the colonies start to peel off and gaps between the cells become visible, remove the EDTA and carefully add 500 microliters of DPBS. Aspirate the DPBS and flush the well with 500 microliters of PSC culture medium using a P-1000 pipette.Pipette up and down to disperse the colonies into clumps of appropriate size. Depending on the confluency, the desired density of the seeded cells and IPSC clonal preference, transfer 1/10 to 1/50 of the cell clump suspension to the new wells. Gently move the plate back and forth several times to distribute the clumps evenly in the well.Incubate the plate for at least 30 minutes at 37 degrees Celsius to let the clumps attach. Change the medium every two to three days until the colonies grow large enough for passaging. After isolation, squamous cells and various smaller round cells can be seen that got excreted with the urine.The first adherent proliferating cells can be seen, and these cells grow into large colonies that can be passaged. Two distinct cell types can be seen, one having an epithelial-like phenotype and the other showing a more mesenchymal-like phenotype with elongated shape. After the first passage, urinary cells grow as a monolayer.During IPSC's generation, some adherent cells can be seen and these cells start to show morphological changes, indicating reprogramming of the cells. Further, the cells that form colonies display the typical embryonic stem cell morphology. All generated IPSCs share the typical embryonic stem cell-like colony morphology, characterized by tightly-packed cells with clearly-defined edges.Immunocytochemistry was used to test the expression of pluripotency associated markers in gorilla IPSCs. Moreover, flow cytometry analysis showed that the analyzed orangutan IPSCs are positive for TRA-1-60. Furthermore, the gorilla IPSCs are able to differentiate into endoderm, mesoderm and ectoderm lineages.An increased number of differentiated cells and a loss of border integrity and uniformity indicate poor IPSC quality. In contrast, defined borders, tight cellular packaging and prominent nucleoli signify high quality IPSCs. Due to the coronal variability, one needs to optimize the clone specific conditions such as splitting ratio, passage timing, and clump size to keep primate IPSCs growing healthy.As quality controls of established IPSCs, one can verify pre-potency by direct or indirect differentiation induction such as in vitro EV differentiation, besides checking marker gene expression.

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Biology

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 ...

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 ...

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.

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 ...

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, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

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 ...

Generation of Human Kidney Tubuloids from Tissue and Urine

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high efficiency. Normal kidney tubuloids recapitulate many aspects of their tissue of origin. They represent distinct nephron segments - most notably of the proximal tubule, loop of Henle, distal tubules, and collecting duct - and can be used to study normal kidney physiology. Furthermore, tubuloid technology facilitates disease modeling, e.g., for infectious diseases as well as for cancer. Obtaining kidney epithelial cells for tubuloid generation is, however, dependent on leftover surgical material (such as partial) nephrectomies) or needle biopsies. The ability to grow tubuloids from urine would provide an alternative, less invasive source of healthy kidney epithelial cells. It has been previously shown that tubuloid cultures can be successfully generated from only a few milliliters of freshly collected urine. This article describes the protocols to generate and propagate ASC-derived human kidney tubuloid cultures from tissue and urine samples.

Human kidney tubuloid cultures represent a valuable in vitro model to study kidney physiology and disease. Tubuloids can be established from kidney tissue (healthy and diseased) as well as urine, the latter representing an easily obtainable and less invasive source of research material.

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high ...