Gli autori desiderano ringraziare per la preziosa collaborazione di: Dott. Neal First e Kourtney Wilkinson per il manoscritto di editing; Dr. Lucy Senter, il Dr. Brigit Willeford e Mike Basett per l&#39;assistenza con cura la formazione e animali presso la struttura del mouse ALAC MSU accreditati; Dr. Dwayne Wise per la sua assistenza con la microscopia e la cattura dell&#39;immagine; Cesar Monroy, Hannah Swoope, e Bobbie Huddleston per la loro assistenza con la produzione video a MSU. Questa ricerca è stata finanziata dall&#39;Ufficio di ricerca istituzionale e il Dipartimento di Scienze Biologiche presso Mississippi State University.

Parte 1: Laparotomia mouse in gravidanza <ol><li> Utilizzando dislocazione cervicale, l&#39;eutanasia un topo gravide C57BL6J femminile a 10,5-11,5 dpc. </li><li> Pulire l&#39;addome con sapone antimicrobico, e poi, radersi. </li><li> Dopo la rasatura, lavare l&#39;addome con una soluzione salina. </li><li> Poi, a secco l&#39;addome con una garza sterile. </li><li> Coprire il mouse con un nuovo campo sterile. </li><li> Effettuare una incisione ventrale con pinze e forbici dissezione. </li><li> Identificare e rimuovere l&#39;utero intero dalla cavità addominale. Embrioni di topo saranno visibili all&#39;interno dell&#39;utero. </li><li> Trasferire l&#39;utero in una scatola Petri riempite con D-PBS, e tenerlo in ghiaccio. </li><li> Usando un bisturi sterile e pinzette, rimuovere la placenta ed extra tessuti embrionali di ogni embrione. </li><li> Trasferire gli embrioni in un piatto fresco Petri riempite con D-PBS. </li><li> Misurare la lunghezza della embrioni di topo. Abbiamo scoperto che le dimensioni differiscono in base all&#39;età dell&#39;embrione. Per esempio 8,5 dpc misurata a una media di 6 millimetri, 10.5 dpc misurata a una media di 11mm, e 12,5 dpc misurato ad una media di 16 mm. </li><li> Quindi, rimuovere la coda per l&#39;estrazione del DNA. </li></ol> Parte 2: dissezione Ridge gonadica Sotto stereomicroscopio e Sorgente Schott Fostec <ol><li> Inserire una carta da filtro in una capsula di Petri. Poi, posto l&#39;embrione del mouse sulla parte superiore del filtro di carta per asciugare l&#39;embrione e immobilizzare per la dissezione. </li><li> Usando un bisturi sterile, praticare un taglio trasversale dell&#39;embrione mouse sopra l&#39;origine del cordone ombelicale. </li><li> Sotto uno stereomicroscopio, utilizzando una pinza sottile sterile e un ago sterile presa in giro fine, rimuovere l&#39;intestino ed il fegato in modo che il sistema urinario è visibile. I reni sono situati lateralmente nella cavità addominale, e la vescica si trova mediale e caudale rispetto ai reni. L&#39;embrione può essere identificato come maschio, perché le creste delle gonadi sono situate su entrambi i lati della vescica. Nella femmina, le creste delle gonadi sono saldamente attaccati alla fine caudale laterale dei reni. </li><li> Sbucciate la cresta gonadica fuori facendo scorrere un ago dietro. La cresta gonadica viene rimosso tagliando via i tessuti che lo supportano. </li><li> Trasferire le creste delle gonadi di un nuovo piatto di Petri riempite con nuove D-PBS. Microscopicamente, la cresta delle gonadi maschili dovrebbero essere spogliati, di grandi dimensioni e di forma ovale. Al contrario, la cresta delle gonadi femminili devono essere individuati, hanno forma allungata ed essere più piccole rispetto alla cresta delle gonadi maschili. </li></ol> Parte 3: Digestione Ridge gonadica Sotto stereomicroscopio: <ol><li> Mesonefro Dissection: Separare i gonade dal mesonefro e la cresta con un ago sottile e tagliente. <ul><li> E &#39;importante rimuovere il mesonefro dalla cresta gonadica, perché è un tessuto somatico che invadere nel processo di derivazione PGC. </li></ul></li><li> Digestione Ridge gonadica: Raccogliere pulito creste gonadico in una goccia di fresca 0.5ul D-PBS. Aggiungere 20 ml di Collagenasi / dispasi soluzione (concentrazione finale a 1mg/ml). <ul><li> Gli embrioni devono essere trattati singolarmente. Ogni embrione deve avere il proprio piatto a parte ed etichettato Petri. </li></ul></li><li> Gonadica Ridge tritare: Tagliare la cresta gonadica in piccoli pezzi con un ago sterile e sterile una pinza sottile No. 4. </li><li> Incubazione: piastre di Petri sono trasferiti in un incubatore a 37 ° C per 15 minuti. <ul><li> Il tempo di incubazione può variare tra i tessuti. </li></ul></li><li> Pipettaggio: Dopo 15 minuti, il tessuto può essere dissociato da pipettaggio. Con pipette in vetro tirato capillari con diametri tra 40 mm -100 Rompere i pezzi pipettando su e giù. Questo formerà grumi di piccole dimensioni. Trasferire le macchie di 0,5 ml di un pre riscaldato D-PBS goccia per un lavaggio finale. </li><li> Dopo il lavaggio, trasferire i grumi di tubi eppendorf. <ul><li> Non più di esporre il tessuto di collagenasi. </li><li> Non fare una sospensione singola cellula. Questo è importante per la formazione di colonie. Se si tratta di una sospensione di cellule singole, PGC tendono a differenziare e migrazione. </li><li> Questo processo non dovrebbe essere più di 20 minuti, o le cellule perdono vitalità. </li></ul></li><li> Centrifugare a 2000 rpm per 5 minuti a temperatura ambiente. </li><li> Dopo centrifugazione, rimuovere il supernatante e risospendere il pellet in 0,5 ml di bussare integrato fuori media (pre-riscaldata a 37 ° C). </li></ol> Parte 4: Primordiale Cultura Germinal Cellule &quot;È necessario effettuare i seguenti passi in fretta per conservare la vitalità di PGC per l&#39;isolamento e la derivazione. <ol><li> 24 ore prima di questo processo si dovrebbe preparare un mitoticamente inattivato MEF-CF1 alimentatore strato seguendo il protocollo di J. Zhang et al 2008. <ul><li> Preparare 20 piatti per uno pregnant femminile del mouse (6-8 embrioni). </li><li> MEF perdono la capacità di essere uno strato alimentatore (promuovere la crescita e prevenire differenziazione) dopo 5-6 passaggi. </li><li> Non utilizzare più di 48 ore MEF. </li></ul></li><li> Immediatamente prima PGC placcatura, il mezzo MEF deve essere lentamente dallo strato alimentatore MEF. </li><li> Quindi, aggiungere 0,5 ml / pozzetto di pre-riscaldato medio knockout integrata sullo strato alimentatore MEF. <ul><li> MEF supporto contiene siero fetale bovino, che induce la differenziazione di PGC. </li><li> Medio knockout deve essere integrato con specifici fattori di crescita specie immediatamente prima dell&#39;uso per assicurare l&#39;attività del fattore di crescita. </li></ul></li><li> Utilizzando distribuzione simmetrica, PGC piatto sopra lo strato alimentatore MEF. <ul><li> Se questi pezzi sono troppo vicini, tendono ad aggregarsi e fare dense colonie che si attaccano male o iniziare differenziazione. </li><li> Piatti di cultura devono essere etichettati con nome embrionali Germinal linea cellulare, il numero di passaggio, e la data. </li></ul></li><li> Cura, il trasferimento i piatti della cultura in un incubatore a 37 ° C e 5% di CO 2. </li><li> Monitorare le cellule ogni 24 ore. </li><li> Dopo 48 ore, togliere 250 ml di mezzi di comunicazione dalla cima della cultura per non disturbare l&#39;attaccamento della colonia. Quindi, aggiungere 250 ml di fresca integrato knock-out dei media (pre-riscaldato a 37 ° C). </li><li> Dopo il punto 7, attendere altre 48 ore, e poi, rimuovere completamente tutti i media e sostituirlo con fresca dei media integrato knock-out (pre-riscaldato a 37 ° C). Fate questo ogni 2 giorni per 8-10 giorni fino a formare PGC colonie. </li></ol> Parte 5: cellule germinali embrionali <ol><li> Dopo 10 giorni di isolamento PGC, colonie cellula germinale embrionale si formano. </li><li> Linee di cellule embrionali germinali sono tenuti in vita da passaggi manuali ogni 8-10 giorni. Essi continuano a presentare una morfologia indifferenziata ed esprimere marcatori pluripotenza come alcalino fosfatasi, Oct-4, SSE-1, e SOX-2 </li></ol> Note <ul><li> Condizioni di sterilità / asettiche sono essenziali nella sala della cultura. </li><li> Per la derivazione e la cultura, PGC sono trattati in classe di sicurezza II purificatore Cabinet Bio. </li><li> Incubazioni sono in un umidificata 37 ° C e 5% CO 2 incubatore. </li><li> Tutti i media ei reagenti vengono filtrati prima di utilizzare in un filtro di 0,2 um, conservato a 4 ° C, e pre-riscaldato a 37 ° C prima dell&#39;uso. </li><li> Durante le fasi di dissezione, tutti gli embrioni e tessuti deve essere tenuta in ghiaccio. </li><li> Tutti i flaconi dei reagenti sono decontaminati con etanolo prima di mettere in un armadio. </li><li> Uso di guanti, camice da laboratorio, e tappi infermiere sono obbligatori. </li></ul> Conclusione Abbiamo fornito un video che mostra come isolare, derivano, e la cultura cellule embrionali germinali da creste delle gonadi di embrioni di topo 10,5-11,5 dpc. L&#39;isolamento riproducibile e la cultura a lungo termine di linee di cellule embrionali germinali fornisce una base fondamentale per lo studio dello sviluppo embrionale precoce, i modelli germinali genetiche ed epigenetiche, la formazione delle gonadi, gli effetti ambientali di organi circostanti durante il processo di sviluppo della gametogenesi negli embrioni maschili e femminili, e la determinazione di percorsi che porta al cancro e la sterilità. <img src="/files/ftp_upload/1635/1635figure1.jpg" alt="Figura 1" />

<ol>
	<li>DeFelici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology: A laboratory Handbook. 1, 73-85 (1998).</li><li>Donovan, P. J., De Miguel, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+Germ+Cells+into+Stem+Cells.">Turning Germ Cells into Stem Cells.</a> Current Opinion in Genetics and Development. 13, 463-471 (2003).</li><li>Labosky, P. A., Hogan, B. L. M., Sharp, P. T., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+12:+Mouse+Primordial+Germ+Cells:+isolation+and+in+vitro+culture.">Part 12: Mouse Primordial Germ Cells: isolation and in vitro culture.</a> Molecular embryology: methods and protocols. 97, (1999).</li><li>Mc Laren, A., Southee, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+Mouse+Embryonic+Germ+Cells+into+Meiosis.">Entry of Mouse Embryonic Germ Cells into Meiosis.</a> Developmental Biology. 187, 107-113 (1997).</li><li>Yoshimizu, T., Obinata, M., Matsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+tissue+and+cell+interactions+play+key+roles+in+mouse+germ+cell+specification.">Stage-specific tissue and cell interactions play key roles in mouse germ cell specification.</a> Development. 128, 481-490 (2001).</li><li>Zhang, J., Hhvorostov, I., Teitell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+MEFs+to+Matrigel+I:+Passaging+hESCs+in+the+Presence+of+MEFs.">From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs.</a> J Vis Exp. , (2008).</li></ol>

Questa ricerca è stata condotta dopo la revisione e la cura degli animali istituzionali e Usa Committee (IACUC) l&#39;approvazione del nostro protocollo del mouse # 08030 (Il ruolo della Reactive Oxygen Species, ROS in fase di sviluppo della linea germinale e la formazione del tumore: Holding e allevamento protocollo) a Mississippi State University.

Pinze n. 14 e 15, bisturi 35 mm, forbici dissezione, campi chirurgici, sapone antisettico, soluzione salina 0,9%, garza, schiuma contenitore di ghiaccio, lama di rasoio, bisturi 35 mm, pinza sottile n ° 4 e 5, con ago sottile presa in giro con maniglia, filtro fogli di carta, e tirò pipette di vetro.<br /<br /&gt; Equipaggiamento: stereomicroscopio da dissezione, sorgente luminosa Schott Fostec, microscopio invertito, micropipetta 10 e 200ul, centrifuga, cappa di sicurezza biologica.

isolation and derivation of mouse embryonic germinal cells

La capacità di cellule embrionali germinali (EG) a differenziarsi in cellule germinali primordiali (PGC) e più tardi in gameti durante le prime fasi di sviluppo è un modello perfetto per affrontare la nostra ipotesi sul cancro e la sterilità. Questo protocollo mostra come isolare le cellule germinali primordiali di gonadi in via di sviluppo 10,5-11,5 giorni post coitum (DPC), embrioni di topo. Lo sviluppo di creste delle gonadi da embrioni di topo (C57BL6J) erano dissociate dalla rottura meccanica con collagenasi, poi placcato in uno strato di fibroblasti di topo alimentatore embrione (MEF-CF1) che precedentemente era mitoticamente inattivati ​​con mitomicina C in presenza di mezzi di comunicazione ad eliminazione diretta e integrata con Inibitore leucemia Factor (LIF), fondamentale fattore di crescita dei fibroblasti (bFGF), e Stem Cell Factor (SCF). L&#39;utilizzo di questi metodi di ottimizzazione per l&#39;identificazione PCG, l&#39;isolamento, e la creazione di condizioni di coltura permette di culture a lungo termine delle cellule EG per più di 40 giorni. Le linee sulle cellule germinali hanno mostrato fenotipo embrionale ed espressione di marcatori comuni usati dello stato pluripotente. L&#39;isolamento e la derivazione di cellule germinali in coltura fornire uno strumento per comprendere il loro sviluppo in vitro e offrono la possibilità di monitorare i danni cumulativi a livelli genetici ed epigenetici dopo l&#39;esposizione allo stress ossidativo.

Watch this Scientific Journal Video about Isolation and Derivation of Mouse Embryonic Germinal Cells at JoVE.com

Isolation and Derivation of Mouse Embryonic Germinal Cells

La capacità di cellule germinali embrionali di differenziarsi in cellule germinali primordiali durante le prime fasi di sviluppo è un modello perfetto per affrontare la nostra ipotesi sul cancro e la sterilità. Questo protocollo mostra come isolare le cellule germinali primordiali di gonadi in via di sviluppo 10,5-11,5 embrioni giorni post coitum mouse.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

isolation-and-derivation-of-mouse-embryonic-germinal-cells

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JoVE Journal

Biology

16.0K Views.  Mississippi State University. La capacità di cellule germinali embrionali di differenziarsi in cellule germinali primordiali durante le prime fasi di sviluppo è un modello perfetto per affrontare la nostra ipotesi sul cancro e la sterilità. Questo protocollo mostra come isolare le cellule germinali primordiali di gonadi in via di sviluppo 10,5-11,5 embrioni giorni post coitum mouse.

Video: Isolation and Derivation of Mouse Embryonic Germinal Cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Ricerca

Biologia

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivazione di cellule staminali embrionali umane da Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Derivazione di cellule staminali trofoblasto mouse da blastocisti

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adulte e embrionali muscolo scheletrico Microexplant Cultura e isolamento di cellule staminali del muscolo scheletrico

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Isolamento e la coltura di cellule embrionali aviaria progenitrici valvolare

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

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 na&iuml;ve 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 <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, 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.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolamento e coltura di cellule dalla Zona nefrogenica del Rene embrionali di topo

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparazione di cellule di fibroblasti embrionali di topo adatto per colture di cellule pluripotenti umane embrionali e staminali indotta

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolamento della embrionali ventricolari cellule endoteliali

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Proteine ​​Isolamento dal Sviluppare embrionali di topo Cuore Valve Regione

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...