Name: application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts
Uploaded: 2014-05-13T23:35:00
Description: Watch this Scientific Journal Video about Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts at JoVE.com

Finansieringen blev leveret af &quot;Progetto en Concorso Fondazione IRCCS Ospedale Maggiore Policlinico 2009-2010&quot; til MD, og ​​Associazione Bambino Nefropatico ABN Onlus, Milano

Alle dyreforsøg blev udført i henhold til de nationale og europæiske gældende regler om beskyttelse af dyr, der anvendes til videnskabelige formål og blev gennemgået og godkendt af den etiske komité på Milanos universitet. 1.. Isolering af Primære Osteoblaster Metoden med mindre ændringer, følger fremgangsmåden beskrevet af Dodig et al 14. <ol><li> Forbered fordøje medium ved tilsætning af 0,1% collagenase P og...

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E., J&#228;hn, K., Prideaux, M., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+primary+osteocytes+from+the+long+bones+of+skeletally+mature+and+aged+mice.">Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice.</a> Biotechniques. 52 (6), 361-373 .</li><li>Kalajzic, I., Matthews, B. G., Torreggiani, E., Harris, M. A., Divieti Pajevic, P., Harris, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+approaches+to+study+osteocyte.">In vitro and in vivo approaches to study osteocyte.</a> Bone. 54 (2), 296-206 .</li><li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+an+osteocyte-like+cell+line,+MLO-Y4.">Establishment and characterization of an osteocyte-like cell line, MLO-Y4.</a> J Bone Miner Metab. 17 (1), 61-65 (1999).</li><li>Mattinzoli, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+in+vitro+osteocytogenesis+induced+by+retinoic+acid+treatment.">A novel model of in vitro osteocytogenesis induced by retinoic acid treatment.</a> Eur Cell Mater. 24, 403-425 (2012).</li><li>Ross, S. A., McCaffery, P. J., Drager, U. C., De Luca, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+in+embryonal+development.">Retinoids in embryonal development.</a> Physiol Rev. 80 (3), 1021-1054 (2000).</li><li>Clagett-Dame, M., McNeill, E. M., Muley, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+all-trans+retinoic+acid+in+neurite+outgrowth+and+axonal+elongation.">Role of all-trans retinoic acid in neurite outgrowth and axonal elongation.</a> J Neurobiol. 66 (7), 739-756 (2006).</li><li>Vaughan, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATRA+induces+podocyte+differentiation+and+alters+nephrin+and+podocin+expression+in+vitro+and+in+vivo.">ATRA induces podocyte differentiation and alters nephrin and podocin expression in vitro and in vivo.</a> Kidney Int. 68 (1), 133-144 (2005).</li><li>Dodig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+TAAT-containing+motif+required+for+high+level+expression+of+the+COL1A1+promoter+in+differentiated+osteoblasts+of+transgenic+mice.">Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice.</a> J Biol Chem. 271 (27), 16422-16429 (1996).</li><li>Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemistry.">Immunocytochemistry.</a> A practical guide for biomedical research. , (2010).</li><li>Woo, S. M., Rosse, r. J., Dusevich, V., Kalajzic, I., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+line+IDG-SW3+replicates+osteoblast-to-late-osteocyte+differentiation+in+vitro+and+accelerates+bone+formation+in+vivo.">Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo.</a> J Bone Miner Res. 26 (11), 2634-2646 (2011).</li><li>Gu, G., Nars, M., Hentune, n. T. A., Metsikk&#246;, K., V&#228;&#228;n&#228;nen, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+primary+osteocytes+express+functional+gap+junctions+in+vitro.">Isolated primary osteocytes express functional gap junctions in vitro.</a> Cell Tissue Res. 323 (2), 263-271 (2006).</li><li>Boukhechba, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+osteocyte+differentiation+in+a+3D+culture+system.">Human primary osteocyte differentiation in a 3D culture system.</a> J Bone Miner Res. 24 (11), 1927-1935 (2009).</li><li>Krishnan, V., Dhurjati, R., Vogler, E. A., Mastro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+in+vitro:+from+pre-osteoblasts+to+osteocytes:+a+contribution+from+the+Osteobiology+Research+Group,+The+Pennsylvania+State+University.">Osteogenesis in vitro: from pre-osteoblasts to osteocytes: a contribution from the Osteobiology Research Group, The Pennsylvania State University.</a> In Vitro Cell Dev Biol Anim. 46 (1), 28-35 .</li><li>Irie, K., Ejiri, S., Sakakura, Y., Shibui, T., Yajima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+mineralization+as+a+trigger+for+osteocyte+maturation.">Matrix mineralization as a trigger for osteocyte maturation.</a> J Histochem Cytochem. 56 (6), 561-567 .</li><li>Hirao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+is+an+important+mediator+of+the+transformation+of+osteoblasts+to+osteocytes.">Oxygen tension is an important mediator of the transformation of osteoblasts to osteocytes.</a> J Bone Miner Metab. 25 (5), 266-276 (2007).</li><li>Brounais, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+oncostatin+M+treatment+induces+an+osteocyte-like+differentiation+on+osteosarcoma+and+calvaria.">Long term oncostatin M treatment induces an osteocyte-like differentiation on osteosarcoma and calvaria.</a> Bone. 44 (5), 830-839 (2009).</li><li>Gupta, R. R., Yoo, D. J., Hebert, C., Niger, C., Stains, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+an+osteocyte-like+phenotype+by+fibroblast+growth+factor-2.">Induction of an osteocyte-like phenotype by fibroblast growth factor-2.</a> Biochem Biophys Res Commun. 402 (2), 258-264 (2010).</li><li>Poole, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sclerostin+is+a+delayed+secreted+product+of+osteocytes+that+inhibits+bone+formation.">Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation.</a> FASEB J. 19 (13), 1842-1844 (2005).</li><li>Dallas, S. L., Prideaux, M., Bonewald, L. 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I de seneste år er osteocyt har vist sig som den mest centrale celle i knoglen. Forskning fremskridt gradvis afslører et antal tidligere uventede eller uprøvede osteocyt egenskaber, som er af enorm værdi til at designe nye og bedre behandling for en række af knoglesygdomme. Imidlertid har undersøgelse af osteocyt biologi blevet ramt af den begrænsede tilgængelighed af in vitro-modeller til en sådan grad, at osteoblaster har været anvendt som surrogat celler over en lang periode inden osteocyt cellelin...

Osteocytter, den mest rigelige knogle celletype, er terminalt differentierede, stærkt forgrenede celler dybt placeret inden skelettet. Cellen kroppen af ​​modne osteocytter er indeholdt i knogle mangler og har forskellige former; osteocytter med aflange celle organer findes i barkbenet, mens afrundede osteocytter er hyppigere hos knogletrabecula 1.. Forgrenede dendritter strækker sig fra cellen kroppen og opholde sig i bittesmå kanaler kaldes canaliculi, danner et indviklet net, der gør flere kontakter, ikke kun med andre osteocytter, men også med andre knogle celletyper, knoglemarv, blodkar og tilhørende pericytes. Gennem interstitielle væske, der er indeholdt i lakuner og canaliculi, osteocytter er også i sidste ende er forbundet til det cirkulerende system, og derfor kan de ikke kun have indflydelse lokale, men også systemiske hændelser, og vice versa deres adfærd kan reguleres af både lokale og systemiske 2 ændringer. Denundersøgelse af osteocytter har for nylig fået momentum, takket være adskillige tekniske fremskridt, såsom generation af vævs-og celle-specifikke transgene dyr, brug af stærke mikroskopi teknikker og af høj-throughput molekylære screening 3,4. Men viden af disse celler er stadig ufuldstændig, hovedsagelig på grund af den relative knaphed på passende in vitro-modeller. Faktisk har osteocytter altid været vanskeligt at opnå og vedligeholde i kultur på grund af den dybe placering, og det lave niveau af spredning, der kendetegner en sådan differentieret celletype. Langs år er der blevet udviklet en række metoder til at isolere primære osteocytter 5-7, selvom de generelt producerer lave celleudbytter og medføre en risiko for, at selv nogle få kontaminerende fibroblaster hurtigt vil overvokse osteocytter 8. Af denne grund, har mest eksperimenterende in vitro arbejde hidtil blevet gennemført på den veletablerede osteocyt celle line MLO-Y4 9. Yderligere in vitro metoder kan øge muligheden for at studere disse celler og kunne forbedre analysen af osteocyt biologi og patofysiologi. Stort set at blive vedtaget, bør sådanne metoder være let at reproducere, og vil ikke kræve specielle instrumenter eller meget lange gange for at nå cellemodning. Vigtigere er det, hvis det er relevant for primærelementer, ville de gøre det muligt at drage fordel af transgene dyr. Vi har for nylig beskrevet, at behandling af osteoblast cellelinien MC3T3-E1 og primære osteoblaster med retinsyre fremkalder en hurtig ændring fænotype, der fører til udviklingen af en homogen population af forgrenede celler, der bærer morfologiske og molekylære egenskaber af osteocytter 10. All-trans-retinoinsyre (ATRA) er en aktiv metabolisk produkt af vitamin A, som regulerer gentransskription ved at binde nukleare retinoinsyrereceptorerne (RAR). RAR&#39;erbinder til DNA som heterodimere med retinoid-X-receptorerne (RXR&#39;er), der førte til modulering af retinsyre (RA)-responsiv målgener 11. ATRA vist sig at modulere differentiering og modning af flere celletyper, heriblandt andre forgrenede celler, såsom neuronale celler 12 og podocytes 13. Metoden beskrives her, er baseret på tilsætning af ATRA til primære osteoblaster. For at være effektiv, ATRA tilføjes på en præcis modning etape celler udpladet ved en defineret tæthed; i vores erfaring disse forhold er afgørende for at opnå den fænotype skifte fra osteoblaster til osteocytter.

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			<td height="20" style="height:20px;width:283px;">Name</td>
			<td style="width:239px;">Company</td>
			<td style="width:219px;">Cat #</td>
			<td style="width:183px;">comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase P</td>
			<td>Roche Applied Science</td>
			<td>11213857001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco, Life Technologies</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco, Life Technologies</td>
			<td>14175129</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alpha-MEM</td>
			<td>Invitrogen, Life Technologies</td>
			<td>22571038</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin/Penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol 2-phosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATRA</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Protect ATRA from light</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Dissolve in PBS and filter before use. Work always under a chemical hood.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>32670</td>
			<td>Can be added to the secondary antibody</td>
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			<td height="20" style="height:20px;">Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Janus Green</td>
			<td>Sigma-Aldrich</td>
			<td>201677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>176745</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorsave</td>
			<td>Calbiochem - Merck</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #7, 0.40 x 0.5mm tips</td>
			<td>World Precision Instruments</td>
			<td>501981</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #4, 0.40 x 0.45mm tips</td>
			<td>World Precision Instruments</td>
			<td>501978</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors, straight,10cm curved</td>
			<td>World Precision Instruments</td>
			<td>14394</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Scissors, 14 cm, straight, S/S</td>
			<td>World Precision Instruments</td>
			<td>501218</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
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			<td height="20" style="height:20px;">Culture flasks</td>
			<td>Corning</td>
			<td>430168</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Well-plates</td>
			<td>Corning</td>
			<td>3335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermanox coverslips</td>
			<td>Thermo Scientific Nunc</td>
			<td>12-565-27</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
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			<td></td>
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			<td height="20" style="height:20px;">microscope</td>
			<td>Zeiss Apotome</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">spectrometer</td>
			<td>Safas Xenius</td>
			<td></td>
			<td></td>
		</tr>
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application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts

<html> Behovet for osteocyt kulturer er velkendt for samfundet i knogle forskere; isolering af primære osteocytter er vanskelig og medfører lave celleantal. Derfor er den mest udbredte cellulært system er osteocyt-lignende MLO-Y4 cellelinie. Fremgangsmåden beskrevet her henviser til brugen af ​​retinsyre til at generere en homogen population af forgrenede celler med morfologiske og molekylære osteocyt funktioner. Efter isolering af osteoblaster fra mus calvaria er all-trans retinoid syre (ATRA) tilsat til celle medium, og celle kontrollen udføres dagligt under et inverteret mikroskop. Første morfologiske ændringer kan påvises efter 2 dages behandling og differentiering er i almindelighed fuldstændig i 5 dage, med den gradvise udvikling af dendritter, tab af evnen til at producere ekstracellulære matrix, nedregulering af osteoblast markører og opregulering af osteocyt-specifikke molekyler. indhold &quot;&gt; er nødvendig Daglig overvågning celle på grund af den iboende variabilitet af primærelementer, og protokollen kan tilpasses med minimal variation celler opnået fra forskellige musestammer og anvendes på transgene modeller. Fremgangsmåden er let at udføre og kræver ikke specielle instrumenter, er meget reproducerbar og hurtigt frembringer et modent osteocyt befolkning i fuldstændigt fravær af ekstracellulær matrix, der tillader anvendelse af disse celler til ubegrænset biologiske anvendelser.

Resultaterne blev opnået fra 5 til 10 uafhængige forsøg. Cellemorfologi AA / GP behandlede primære celler har det meste brosten-lignende funktioner, karakteristisk af modne osteoblaster. Afbrudt forgrenede celler (som angivet med røde pile i figur 1A) kan findes, som sandsynligvis repræsenterer nogle osteocytter. Med ATRA behandling, celler hurtigt begynde at vise forgreninger, der normalt observeres efter 2 dage. Som...

Watch this Scientific Journal Video about Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts at JoVE.com

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Anvendelse af retinsyre hentes osteocytter kulturer fra Primary Mouse Osteoblaster

Behandling af primære muse osteoblaster med retinsyre producerer en homogen population af forgrenede celler, der bærer morfologiske og molekylære egenskaber af osteocytter. Fremgangsmåden overvinder vanskeligheden ved at opnå og bevare primære osteocytter i kultur og kan være fordelagtigt at undersøge celler afledt fra transgene modeller.

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

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Biology

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

Generering af mus afledt fra induceret pluripotente stamceller

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

Isolering af primær melanocytstimulerende lignende celler fra mus Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

NaÃƒÆ’Ã‚Â¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Naive voksne stamceller Isolering fra primære humane fibroblastkulturer

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Isolering af Primære myofibroblasts fra mus og Human tyktarmsvæv

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Fremstilling af primær Myogenic precursorcelle / myoblast kulturer fra Basal hvirveldyr afstamninger

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...