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.