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.

The authors have no conflicts of interest to disclose.

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><tbody><tr><td>Accumax&amp;trade; cell detachment solution (&lt;em&gt;Detachment solution&lt;/em&gt;)</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&amp;nbsp;</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&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-122-965</td><td>Dilution: 1/50</td></tr><tr><td>Bambanker&amp;trade; (&lt;em&gt;Cell freezing medium&lt;/em&gt;)</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&amp;trade; II automated cell counter</td><td>Thermo Fisher Scientific</td><td>AMQAX1000</td><td></td></tr><tr><td>CryoKing&amp;reg; 1.5 mL Tubes with 2D Barcode (&lt;em&gt;Cryotubes&lt;/em&gt;)</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&amp;trade; EmGFP Sendai Fluorencence Reporter (&lt;em&gt;GFP Sendai virus&lt;/em&gt;)</td><td>Thermo Fisher Scientific</td><td>A16519</td><td></td></tr><tr><td>CytoTune&amp;trade;-iPS 2.0 Sendai Reprogramming Kit (&lt;em&gt;Sendai virus reprogramming kit&lt;/em&gt;)</td><td>Thermo Fisher Scientific</td><td>A16518</td><td></td></tr><tr><td>DAPI 4&#039;,6-Diamidine-2&#039;-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&amp;trade; 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&amp;trade; 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&amp;nbsp;</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&amp;trade; 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&amp;trade; Supplement</td><td>Thermo Fisher Scientific</td><td>35050038</td><td></td></tr><tr><td>Heracell&amp;trade; 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&amp;nbsp;</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&amp;amp;D Systems</td><td>MAB1368</td><td>Dilution: 1/100</td></tr><tr><td>Mouse anti-alpha-Smooth Muscle Actin antibody</td><td>R&amp;amp;D Systems</td><td>MAB1420</td><td>Dilution: 1/100</td></tr><tr><td>Mouse anti-beta-III Tubulin antibody</td><td>R&amp;amp;D Systems</td><td>MAB1195</td><td>Dilution: 1/100</td></tr><tr><td>mTeSR&amp;trade; 1</td><td>STEMCELL Technolgies</td><td>85850</td><td></td></tr><tr><td>Nanog (D73G4) XP Rabbit mAb&amp;nbsp;</td><td>Cell Signaling Technology</td><td>4903S</td><td>Dilution: 1/400</td></tr><tr><td>Normocure&amp;trade; (&lt;em&gt;Antimicrobial Reagent&lt;/em&gt;)</td><td>Invivogen</td><td>ant-noc</td><td></td></tr><tr><td>Oct-4 Rabbit Antibody&amp;nbsp;</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 &amp;micro;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&amp;nbsp;</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&amp;reg; 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&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T8787-50ML</td><td></td></tr><tr><td>TrypLE&amp;trade; Select Enzyme (1x), no phenol red (&lt;em&gt;Dissociation enzyme&lt;/em&gt;)</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 (&lt;em&gt;Rock Inhibitor&lt;/em&gt;)</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 - Day 5)....

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 ...

generation-maintenance-primate-induced-pluripotent-stem-cells-derived

Research

JoVE Journal

Biology

3.3K Views.  Ludwig Maximilian University of Munich. IPSC's van primaten zijn nuttig, maar vaak moeilijk te verkrijgen vanwege ethische en technische kwesties. Dit protocol biedt een zo toegankelijk mogelijk pad voor het genereren en onderhouden van IPSC's van primaten. Het gebruik van urinecellen als primaire bron van IPSC's heeft een groot voordeel omdat ze op een volledig niet-invasieve manier kunnen worden verkregen zonder speciale technieken.De procedure wordt gedemonstreerd door Jessica Radmer, een promovendus van Wolfgang M.S.Labo...

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

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Developmental Biology

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Medicine

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Neuroscience

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

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 and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

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