और भ्रूण माउस किडनी की nephrogenic क्षेत्र से कक्ष के अलगाव और संस्कृति
Summary
इस रिपोर्ट में हम भ्रूण माउस कि संकेत विकासशील गुर्दे के स्टेम / पूर्वपुस्र्ष कोशिकाओं विनियमन रास्ते का अध्ययन करने के लिए इस्तेमाल किया जा सकता है गुर्दे से पूर्वपुस्र्ष सेल आला के अलगाव और संस्कृति के लिए एक विधि का वर्णन. इन संवर्धित कोशिकाओं को अत्यधिक छोटे अणु और पुनः संयोजक प्रोटीन उपचार के लिए पहुंच रहे हैं, और महत्वपूर्ण बात यह भी वायरल पारगमन है, जो उम्मीदवार रास्ते के कुशल हेरफेर की अनुमति देता है.
Abstract
Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering naïve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated http://www.gudmap.org/, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.
Protocol
Discussion
इस प्रोटोकॉल में हम एक विधि को अलग और संस्कृति भ्रूण गुर्दे की nephrogenic क्षेत्र से कोशिकाओं का वर्णन. यह शुरू में nephrogenic क्षेत्र (रिक्त एट अल., 2009) की कोशिकाओं पर BMP7 उपचार के प्रभाव के अध्ययन के हिस्से के रूप म?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस काम DK078161 R01 NIDDK (LO), अमेरिकन हार्ट एसोसिएशन (एबी), स्वीडन और Kungliga Fysiografiska Sällskapet, लंड, स्वीडन (यूबी) के Wenner GREN मूलाधार से postdoctoral फैलोशिप से समर्थित किया गया था. अतिरिक्त समर्थन ऊतकविकृतिविज्ञानी, जैव सूचना विज्ञान और FACS के लिए मेन मेडिकल सेंटर अनुसंधान संस्थान कोर सुविधाएं (2P20RR18789-06 द्वारा समर्थित) और MMCRI पशु सुविधा द्वारा प्रदान किया गया.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Watchmaker’s forceps #5 | Roboz | RS-4905 | 2 pairs required | |
| Collagenase A | Roche | 10 103 578 001 | Wear mask | |
| Porcine Pancreatin | Sigma | P8096 | Wear mask | |
| DPBS w/ Ca, Mg | Lonza | 17-513F | With Mg & Ca | |
| Fetal bovine serum (FBS) | BioWhittaker | 14-901E | ||
| HBSS | Gibco | 14175 | ||
| KSFM | Gibco | 10724-011 | without added growth factors | |
| L-Glutamine 200mM | Sigma | G7513 | Use @ 1% v/v | |
| PenStrep 10,000 U penicillin / ml 10 mg streptomycin / ml | Sigma | P4333 | Use @ 1% v/v | |
| Fibronectin | BD Biosciences | 356008 | Supplied as powder | |
| 24 well culture plate | Thermo Scientific | 142485 | Nunclon | |
| DPBS w/o Mg & Ca | Lonza | 17-512F | ||
| 5mL polystyrene tubes | BD Biosciences | 352235 | ||
| DNase (1 U/μl) | Invitrogen | 18068-015 | 4 μl per 1.5 ml | |
| 40 micron cell-strainer | BD Biosciences | 352235 | w/cap and tube | |
| Paraformaldehyde | Sigma | P6148 | Use @ 4 % w/v | |
| Triton X100 | VWR | VW3929-2 | Use @ 0.3 % v/v | |
| PBS | Sigma | P3813 | Supplied as powder | |
| Donkey serum | Jackson ImmunoResearch | 017-000-121 | Use @ 5 % v/v | |
| Rabbit anti-PAX2 | Invitrogen | 71-6000 | Dilute 1:100 | |
| Rabbit anti-β-gal | MP Biomedicals | 559762 | Dilute 1:200 | |
| Oregon Green 488 phalloidin | Invitrogen | O7466 | Dilute 1:200 | |
| Donkey anti-rabbit Alexa Fluor 568 | Invitrogen | A10042 | Dilute 1:200 | |
| DAPI | Invitrogen | D1306 | Dilute 1:5000 | |
| Vectashield | Vector Laboratories | H-1000 | ||
| Glycerol | EMD | GX0185-6 | Mix 50 % with H2O | |
| RNeasy Micro Kit | Qiagen | 74004 | Use “DNase on column” protocol | |
| Transfer pipettes, 3ml | BD Falcon | 357575 |
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