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

Podsumowanie

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.

Streszczenie

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 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.

Wprowadzenie

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 relevan....

Protokół

This experimental procedure was approved by the responsible ethic committee on human experimentation (20-122, Ethikkommission LMU München). 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 using sterile technique in a biological safety cabinet. All buffer and media compositions can be found in Supplementary Table S1. Ensure....

Wyniki

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 - Day.......

Dyskusje

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.......

Materiały

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