Name: promotion of survival and differentiation of neural stem cells with fibrin and growth factor cocktails after severe spinal cord injury
Uploaded: 2014-07-27T14:00:00
Description: Watch this Scientific Journal Video about Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury at JoVE.com

Vi takker Rat Resource og Research Center, University of Missouri, Columbia, Missouri, for at give GFP rotter; Neuralstem Inc. til at tilvejebringe humane neurale stamceller. Dette arbejde blev støttet af Veterans Administration, NIH (NS09881), Canadian Spinal Research Organization, The Craig H. Neilsen Fonden samt Bernard og Anne Spitzer Charitable Trust.

Alle animalske protokoller godkendes af VA-San Diego Institutional Animal Care og brug Udvalg (IACUC). NIH retningslinier for laboratoriedyr pleje og sikkerhed nøje. Dyrene har fri adgang til mad og vand hele studiet og er tilstrækkeligt behandlet for at minimere smerte og ubehag. 1. Fremstilling af fibrin komponenter indhold vækstfaktor Cocktails <ol><li> 25 mg rotte fibrinogen opløses i 0,5 ml PBS til opnåelse af 50 mg / ml 2x stamopløsning (se Materialer tabel). Fibrinogen er vans...

<ol>
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En af de store forhindringer for NSC transplantation i såret rygmarven er dårlig overlevelse i læsionen center. Eventuelle huller eller hulrum i læsionssted potentielt kunne reducere eller svække dannelsen af ​​funktionelle neuronale relæer mellem supraspinale axoner og adskilt rygmarv segmenter under skaden. Desuden kan ringe overlevelse af podede NSC påvirke deres integration med værtsvæv, og derfor reducere tilslutning af podede neuroner med værten neuroner. Potentielle mekanismer til at forklare celle ...

Rygmarvsskade (SCI) ofte skader, der ikke kun hvide substans skrifter, der bærer signaler til og fra hjernen, men også den centrale grå stof, der forårsager segmentær tab af interneuroner og motoriske neuroner. Konsekvensen af ​​SCI er tab af både motorisk og sensorisk funktion under læsionen. Desværre er den voksne centralnervesystemet (CNS) ikke spontant regenerere, hvilket resulterer i permanente funktionelle mangler 1. Derfor rekonstruktion af den skadede voksne rygmarven og forbedring i motoriske, sensoriske og autonome funktion er et vigtigt mål for SCI forskning. Neurale stamceller (NSC), enten direkte isoleret fra embryonale eller voksne CNS, er overbevisende kandidat celler til at erstatte tabte neuroner og glia. Desuden er disse celler har evnen til at danne nye funktionelle relæer genoprette axonal conduction over en læsionsstedet 2,3. Til dato har der ikke været en detaljeret belysning af anatomiske, electrophysiological og adfærdsmæssige effekter af neuronal relæ dannelse af transplanteret NSC efter alvorlig SCI. Der er flere grunde til dette: For det første, transplanterede NSC eller føtal CNS væv overleve dårligt når de er podet i store læsioner hulrum. Tidligere undersøgelser viser betydelige tab tidligt celle, der forlader store tomme cystisk læsion hulrum 4,5. I nogle studier vil de podede celler efterfølgende dele sig og fylde læsionen hulrum 4,5, men dette kan ske efter en forsinkelse på dage til uger, og efterfølgende læsionssted påfyldning muligvis ikke være komplet eller konsekvent. For det andet blev et effektivt tracking system, der giver pålidelige oplysninger om cellulær overlevelse, differentiering / modning, og udvækst af transplanteret NSC mangler. De fleste tidlige undersøgelser udnyttet antegrad og retrograd mærkning at spore axonale fremskrivninger fra transplantationer 2,3. Men disse teknikker kun delvist og ofte utydeligt mærket axonale fremskrivninger skyldes podede celler og tracer metoder er subject artefakter forårsaget af farvestoflækage over de implanterede celler. Andre grupper brugte menneskelige specifikke neuronale markører til at mærke axonale fremskrivninger efter transplantation af humane føtale NSC i såret gnaver rygmarv 5,6. Men i disse studier xenotransplantaterne ikke konsekvent overleve godt. For nylig blev viral levering af GFP-reportergenet anvendes til at mærke dyrkede NSC 7,8. Men GFP udtryk var ofte usammenhængende og kan nedreguleret 7. For nylig anvendelsen af transgene donor mus eller rotter, der stabilt udtrykker reportergenet, GFP eller human placental alkalisk phosphatase, har forbedret sporing af transplanterede neurale stamceller / stamceller in vivo 9,11. For det tredje, flere undersøgelser tyder på, at in vitro-dyrkede rotte NSC udvundet af enten embryonale eller voksne CNS udelukkende differentiere i gliøse slægter når transplanteres ind i miljø i intakte eller tilskadekomne voksen spinal cord 7,12,13, trods det faktum at disse neurale stamceller er i stand til at differentiere til både neuroner og glia in vitro, hvilket indikerer, at det lokale miljø kan diktere skæbne stamceller. Alternativt kan kulturperler NSC, især dem, der stammer fra voksne CNS, har iboende defaulty egenskab til at differentiere til gliøse slægter in vivo 13. På grund af de begrænsninger diskuteret ovenfor, vores gruppe for nylig udviklet en ny protokol til forbedring af embryonale NSC tracking, overlevelse og differentiering / modning i de alvorligt tilskadekomne voksne rygmarven. Kort, vi begyndte med en stabil transgen Fischer 344 rotte indavl der udtrykker et GFP reporter gen, der opretholder GFP udtryk efter in vivo transplantation 14. Dernæst brugte vi frisk isolerede NSC fra embryonale dag 14 Fischer 344 rygmarv, et udviklingstrin, der bevarer potentiale til at generere både neuroner og glia. Endelig har vi indlejretfrisk dissocierede NSC i en fibrin matrix indeholdende vækstfaktorer 15-17 at fastholde cellerne og jævnt distribuere dem i en stor læsion hulrum, der sigter på at støtte graft celle overlevelse, differentiering og integration. Transplantater blev placeret i lokaliteter af T3 komplet transektion to uger efter rygmarvsskade. Disse transplanterede celler konsekvent fyldt komplet transection sites og differentieret i rigelige neuroner, der udvidede stort antal axoner i vært rygmarven over lange afstande 18. Lignende resultater blev opnået ved anvendelse af dyrkede neurale stamceller grafts humane til immuno-deficiente rotter 18.

<table border="1">
 <tbody>
 <tr>
 <td>Reagents</td>
 <td>Company</td>
 <td>Catalogue</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Fibrinogen(rat)</td>
 <td>Sigma</td>
 <td>F6755-25MG</td>
 <td>2 hr at 37 &deg;C to dissovle 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Thrombin (rat)</td>
 <td>Sigma</td>
 <td>T5772-100UN</td>
 <td>Dissovle in 10 mM CaCl2 
 Stock Concentration: 50 U/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>bFGF (human)</td>
 <td>Sigma</td>
 <td>F0291 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>EGF (murine)</td>
 <td>Sigma</td>
 <td>E1257 (0.1 mg)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>BDNF(human)</td>
 <td>Peprotech</td>
 <td>452-02 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>NT-3 (human)</td>
 <td>Peprotech</td>
 <td>452-03 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>GDNF (rat)</td>
 <td>Sigma</td>
 <td>G1401 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>IGF-1(mouse)</td>
 <td>Sigma</td>
 <td>I8779 (50 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>aFGF (human)</td>
 <td>Sigma</td>
 <td>F5542 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l </td>
 </tr>
 <tr>
 <td>PDGF-AA (human)</td>
 <td>Sigma</td>
 <td>P3076 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>HGF (human)</td>
 <td>Sigma</td>
 <td>H9661 (5 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>MDL28170</td>
 <td>Sigma</td>
 <td>M6690 (25 mg)</td>
 <td>Stock in DMSO 
 Stock Concentration: 1 mM 
 Final Concentration: 50 &mu;M</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Millipore</td>
 <td>BSS-1005-B</td>
 <td>Stock Concentration: 1X 
 Final Concentration: 1X</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Putney</td>
 <td>26637-411-01</td>
 <td>40-80 mg/kg 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Lloyd</td>
 <td>0410,</td>
 <td>2.5-8 mg/ml 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 1.3 mg/ml</td>
 </tr>
 <tr>
 <td>Acepromazine</td>
 <td>Butler</td>
 <td>003845</td>
 <td>0.5-4 mg/ml 
 Stock Concentration: 10 mg/ml 
 Final Concentration: 0.25 mg/ml</td>
 </tr>
 <tr>
 <td>Betadine</td>
 <td>Healthpets</td>
 <td>BET16OZ</td>
 <td></td>
 </tr>
 <tr>
 <td>Ringers</td>
 <td>Abbott</td>
 <td>04860-04-10</td>
 <td>2-3 ml/inj</td>
 </tr>
 <tr>
 <td>Banamine</td>
 <td>Schering-Plough</td>
 <td>0061-0851-03</td>
 <td>2.5 -5 mg/kg 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 0.5 mg/ml</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Sandoz</td>
 <td>0781-3404-85</td>
 <td>80-100 mg/kg 
 Final Concentration: 50 mg/ml</td>
 </tr>
 <tr>
 <td>LH-RH</td>
 <td>Sigma</td>
 <td>L4513</td>
 <td>200 &mu;g/kg 
 Final Concentration: 200 &mu;g/ml</td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Gibco</td>
 <td>14175-096</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Gibco</td>
 <td>25200056 </td>
 <td>Stock Concentration: 0.25 % 
 Final Concentration: 0.125 %</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>11995073 </td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Gibco</td>
 <td>16000044 </td>
 <td>Stock Concentration: 100 % 
 Final Concentration: 10 %</td>
 </tr>
 <tr>
 <td>Neurobasal Medium</td>
 <td>Gibco</td>
 <td>21103049 </td>
 <td></td>
 </tr>
 <tr>
 <td>B27</td>
 <td>Gibco</td>
 <td>17504044</td>
 <td>Stock Concentration: 100x 
 Final Concentration: 1x</td>
 </tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Note, use human reagents for grafts of human NSCs</td>
 </tbody>
</table>

promotion of survival and differentiation of neural stem cells with fibrin and growth factor cocktails after severe spinal cord injury

Neurale stamceller (NSC), kan selv forny og differentiere i neuroner og glia. Transplanterede NSC kan erstatte mistede neuroner og glia efter rygmarvsskade (SCI), og kan danne funktionelle relæer til at re-forbinde rygmarv segmenter over og under en læsion. Tidligere undersøgelser grafting neurale stamceller er blevet begrænset af ufuldstændig transplantatoverlevelse i rygmarven læsion hulrum. Endvidere sporing af transplantat celleoverlevelse, differentiering og proces forlængelsen var ikke blevet optimeret. Endelig i tidligere undersøgelser, dyrkede rotte NSC blev typisk rapporteret at differentiere til glia når podet til de sårede rygmarv, snarere end neuroner, medmindre skæbne blev drevet til en specifik celletype. For at løse disse problemer, har vi udviklet nye metoder til at forbedre overlevelsen, integration og differentiering af NSC til steder med selv svær SCI. NSC var frisk isoleret fra embryonale dag 14 rygmarv (E14) fra en stabil transgen Fischer 344 rotte linje udtrykker grøn fl, fluorescerende protein (GFP) og blev indlejret i fibrin matrix indeholdende vækstfaktorer; denne formulering har til formål at fastholde podede celler i læsionen hulrum og understøtte celleoverlevelse. NSC i fibrin / vækstfaktor cocktail blev implanteret to uger efter, thorax niveau-3 (T3) komplette rygmarvsskader transections og derved undgå spidsbelastningsperioder på inflammation. Resulterende podninger helt fyldt læsionen hulrum og differentieret i både neuroner, som udvidede axoner i værten rygmarven løbet bemærkelsesværdigt lange afstande, og glia. Transplantater af dyrkede humane NSC udtrykker GFP resulterede i lignende fund. Således er metoderne defineret til forbedring af neural stamcelle podning, overlevelse og analyse af in vivo resultater.

GFP immunhistologisk mærkning viser, at transplanterede rotte NSC resuspenderet i phosphatbufret saltvand (PBS) (der mangler en fibrinmatrix og vækstfaktorer), overlevede dårligt i T3 transection site, kun er knyttet til læsionen / vært margin og forlader det meste af læsionsstedet tomt uden transplanterede celler (figur 2A). Overlevelsen af ​​NSC forbedret, når co-podet med fibrin-matricer alene, men transplantatet ikke fuldstændigt at fylde en stor læsion hulrum (data ikke vist). Derfor h...

Watch this Scientific Journal Video about Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury at JoVE.com

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Fibrin matricer indeholdende vækstfaktorer blev brugt til at tilbageholde påhæftede neurale stamceller i lokaliteter af komplette rygmarv transection. Transplanterede celler helt fyldt læsionen hulrum og differentieres i flere neurale celletyper, herunder neuroner, der udvidede axoner i værtscelle rygmarven over lange afstande.

Fremme af overlevelse og differentiering af neurale stamceller med fibrin og Growth Factor Cocktails efter Svær Rygmarvsskader

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Isolering og dyrkning af neurale kamceller fra embryonale Murin Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Komplet Rygmarvsskader og Brain Dissektion Protokol for Efterfølgende whole- mount<em&gt; In Situ</em&gt; Hybridisering i Larver havlampret

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

En<em&gt; Ex vivo</em&gt; Laser-induceret Rygmarvsskader model til opgørelse Mekanismer axon Degeneration i realtid

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging neurale aktivitet i den primære somatosensoriske Cortex ved hjælp af Thy1-GCaMP6s Transgene mus

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

En Neurosphere analyse til at vurdere endogene neurale stamceller aktivering i en musemodel af Minimal rygmarvsskade

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Identifikation af celleoverflade markører for primær neurale stamme og stamceller ved metabolisk mærkning af Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...