Name: isolation and expansion of adult canine hippocampal neural precursors
Uploaded: 2016-11-29T15:00:00.000+00:00
Duration: 9 min 37 s
Description: Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

This work was supported by the National Health and Medical Research Council (NHMRC) of Australia grants (#568969 and 1004152).

뉴 사우스 웨일즈, 호주 법에 따라 사후 부검의 뇌 조직은 연구와 관련이없는 이유로 안락사 성인 개에서 인수되었다. 문화 매체의 1. 준비 <ol><li> 100 ml의 증류수에 젤라틴 분말 0.1 g을 첨가하고, 용해 될 때까지 37 ℃에서 교반하여 0.1 % 젤라틴 용액을 준비한다. 15 분 동안 UV 조사에 의한 살균 용액. 이 매체는 1개월까지 4 ° C에 저장할 수 있습니다. </li><li> DMEM 500 ㎖ (4.5 g / L의 D-포도당)로 F-12의 167 ml에 추가하여 둘 베코의 수정 독수리 중간 (DMEM) 및 영양 혼합 F12의 : 1 비율 3를 준비합니다. (1 % 최종 농도) 페니실린 / 스트렙토 마이신 솔루션의 6.7 ML을 추가합니다. 이 매체 1 개월까지 4 ℃에서 어둠 속에서 저장 될 수있다. </li><li> DMEM (4.5 g / L D- 글루코스), 440 mL의 조합으로 혈청 배지 500 ml를 준비 5 ml의 L- 알라 닐 -L- 글루타민 디 펩티드 (200 mM)을 페니실린 / 스트렙토 마이신 5 ㎖ 태아 bovin 50㎖의즉 혈청 (FBS). 이 매체 1 개월까지 4 ℃에서 어둠 속에서 저장 될 수있다. </li><li> 완전 성장 배지는 줄기 세포의 신경 구성 (NSC) 기본 배지 증식 신경 보충-A (NS-A), 2 % FBS, 2 μg의 / ㎖ 헤파린, 20 ng / ml의 상피 세포 성장 인자 (EGF), 10 ng의 / ㎖ 염기성 섬유 아세포 성장 인자 (bFGF). 수욕에서 37 ° C이 매체는, 신선한 뇌 해부 직전에, 따뜻한합니다. </li></ol> 2. 뇌 추출 <ol><li> 포비돈 - 요오드를 사용하여 강아지의 머리의 등쪽 표면을 살균하여 6 시간의 사후 내에서 추출을 시작합니다. </li><li> 메스를 사용하여 기본 턱 근육을 노출, 두개골의 전체 지느러미 표면을 가로 질러, 시상 중간 선을 따라 세로 절개를합니다. 이 컷의 길이는 강아지의 종 및 연령에 따라 달라집니다. </li><li> 두개골의 주동이의와 꼬리 국경에서 추가로 5 - - 양쪽에 10cm 수직 컷 통과각각 눈과 귀 뒤에 보내고는 피부가 충분히 반영 될 수 있도록합니다. </li><li> 메스를 이용하여 두개골의 시상 크레스트 그들의 첨부의 턱 근육을 분리하고, 두개의 남아있는 결합 조직을 긁어. </li><li> 발진 뼈를 사용하여 원주 라인에 두개골의 캡을 절단 보았다. 프로브를 사용하거나 경질에 남아있는 첨부 파일을 절단 메스 다음 조심스럽게 두개골 캡을 제거하면 뇌의 등쪽 표면을 노출합니다. 주의 : 뼈의 두께는 두개골에서 다른 지역에서 다르므로주의가 너무 깊이 침투하여 기본 뇌에 손상을주지하기 위해 행사해야합니다. </li><li> 상부 경추 사이의 메스를 삽입하여 척수 절단. </li><li> 부드럽게 머리를 들어 올려 한 손으로 조심스럽게 두개골 베루스 포 사이 협곡에서 뇌를 확보하고 복부 측면에있는 연결하는 신경과 혈관을 절단하기 위해 메스를 사용합니다. 조심스럽게 B를 해제두개골의 출력 및 DPBS의 용기에 비. 주의 : 개 뇌의 큰 후각 전구는 앞쪽 뇌 포사에 깊은 연장 할 수있다. 뇌를 제거 할 때 이러한 구조를 유지하기 위해주의를 기울여야한다. </li></ol> 3. 해마 해부 <ol><li> 150mm 배양 접시에있는 혈액을 제거하고 아래 지느러미 측면을 배치하는 둘 베코 인산염 완충 생리 식염수 (DPBS)의 뇌를 씻어. </li><li> 천천히 젖은 뇌 칼 블레이드의 전체 길이를 이용하여 하나의 슬라이스를 이용하여 중앙 시상면을 통해 종 방향으로 머리를 이등분. 추가 하락 압력을 적용하거나이 조직에 손상을 줄 수 등의 절단 동작을 사용하지 마십시오. </li><li> 각 반구의 내측은, 최대 표면 메스 미세 핀셋을 이용하여 해마를 해부하고, 35mm 조직 배양 접시에 옮긴다 배치. </li></ol> 신경 전구 세포의 분리 4. <ol><li> t을 말하다약 1mm 3 조각으로 메스 블레이드를 사용하여 문제 샘플. </li><li> , 15 ㎖의 튜브에 조직을 이동시켜, 0.1 % 트립신 EDTA 2 mL를 넣고 37 ℃ 수조에서 7 분 동안 배양한다. </li><li> 튜브 혈청 배지 4㎖를 첨가하여 효소 반응을 중지. 7 분 100 XG에서 원심 분리하여 현탁액을 펠렛과 뜨는을 제거합니다. </li><li> DPBS 300 μL에 펠렛을 재현 탁시키고, 기계적으로 부드럽게 부드러운 균질이 형성 될 때까지, 1000 μL 피펫 팁을 사용하여 상하로 피펫 팅에 의해 해리. </li><li> 튜브에 혈청 미디어의 14 ML을 추가하고 40 μm의 셀 스트레이너를 통해 세포 현탁액을 전달합니다. 7 분 동안 100 XG에서 원심 분리하여 현탁액을 펠렛 뜨는을 제거하고 전체 성장 매체에 재현 탁. </li></ol> 5. Neurosphere 문화 <ol><li> 분리로 얻어진 세포 현탁액에서 트립 판 블루 염색 배제 및 과다를 사용하여 생존 세포의 수를 계산사이토. </li><li> 6- 웰 플레이트의 웰을 코팅에 1 × 105 세포 / cm 2의 완전한 성장 배지와 시드의 세포 현탁액을 희석. </li><li> 37 ° C (14), 5 % CO 2&gt; 95 % 습도에서 품어 - 28 일 7 일마다 성장 미디어의 50 %를 교체합니다. neurosphere를 정기적의 직경을 측정한다. 직경 100 μm의를 초과 할 수있는 경우, 세포 사멸 및 자연 분화는 neurosphere를 내 발생할 수 있습니다. </li></ol> 6. Neurosphere 항로 <ol><li> neurosphere를 약 100 μm의 직경에 도달 부동 문화, 같은 통로에 하나의 튜브에 모든 neurosphere 포함 된 미디어를 결합한다. 7 분 100 XG에서 원심 분리하여 펠렛과 뜨는을 제거합니다. </li><li> 0.1 % 트립신 EDTA 1 ㎖에 펠렛을 재현 탁하고, 37 ℃에서 수조에 7 분 동안 배양한다. 부드럽게 피펫 100 배의 아래, 200 μL 피펫 팁을 사용 dissociati 있도록neurosphere를의에. </li><li> 7 분 동안 100 XG에 튜브 원심 혈청 배지 5 ㎖를 첨가하여 효소 반응을 중지. </li><li> 상층 액을 분리 한 용액에 세포 펠렛 완전 성장 배지를 재현 탁하고 트리 판 블루 염색 배제과 혈구를 사용하여 생존 세포의 수를 계산. </li><li> 6- 웰 플레이트의 웰을 코팅에 1 × 105 세포 / cm 2의 완전한 성장 배지와 시드의 세포 현탁액을 희석. 7 일마다 성장 배지의 50 %를 교체 28 일 - 37 ° C, 14, 5 % CO 2 및&gt; 95 %의 습도에서 인큐베이션. </li></ol> 7. 자기편 문화 신경 전구체 세포 <ol><li> 종래 접착 배양 neurosphere를 처리로 1 시간 동안 37 ℃의 배양기에서 조직 배양 접시 경화의 표면을 커버하기 위해 0.1 % 젤라틴 용액의 충분한 양을 추가한다. </li><li> 부동 neurosphere 문화에서 하나의 튜브에 모든 미디어를 결합한다. neur 펠렛100 XG에 원심 분리하여 ospheres 7 분 및 뜨는을 제거합니다. </li><li> 0.1 % 트립신 EDTA 1 ㎖에 펠렛을 재현 탁하고, 37 ℃에서 수조에 7 분 동안 배양한다. 부드럽게 피펫과 100 배 아래로, 200 μL 피펫 팁을 사용. </li><li> 7 분 100 XG에 효소 반응과 원심 분리기를 중단 혈청 미디어의 5 ML을 추가합니다. 뜨는을 취소하고 전체 성장 매체 1 ML에있는 세포 펠렛을 재현 탁하고 트리 판 블루 염색 제외하고 혈구를 사용하여 가능한 세포의 수를 계산합니다. </li><li> 완전 성장 배지에서 세포 현탁액을 희석하여 조직 배양 플라스크에서 젤라틴을 제거하고 / cm (2) 1 × 104 세포의 세포를 시드. </li><li> 37 ° C, 5 % CO 2 및&gt; 95 %의 습도에서 세포를 인큐베이션. 문화가 약 ​​80 %의 합류 (후 약 7 일)에 도달 할 때까지 3 일마다 신선한, 예열 완료 성장 매체와 매체를 교체합니다. </li></ol> 8. Neur알 콜로니 형성 분석 <ol><li> 신경 콜로니 형성 세포 (NCFC) 분석의 미디어 구성 요소를 조합 해 NCFC 혈청 배지 증식 NS-A, 콜라겐 용액 (3 ㎎ / ㎖). / ㎖ 헤파린 2 μg의 20 NG / ㎖ EGF, 10 NG / ㎖의 bFGF와 함께이 미디어를 보충합니다. </li><li> 분석 배지 콜로니를 형성하는 신경 세포의 해마 조직 해리 재현 탁 후에, 수욕에서 37 ℃로 예열. 35 mm dish에 1.1 × 105 세포 / ml의 밀도로 클론 세포 종자. </li><li> 37 ° C, 5 % CO 2 및&gt; 95 %의 습도에서이 반고체 콜라겐 매트릭스 세포를 인큐베이션. 이십팔일위한 배양 7 일마다 성장 배지의 50 %를 교체. </li></ol> 신경 전구 세포의 분화 9. <ol><li> differentia 위해 세포를 가공하기 전에 24 시간 동안 37 ℃의 배양기에서 조직 배양 접시 경화의 표면을 덮도록 5 μg의 / cm 2 라미닌 용액의 충분한 양을 추가기. 참고 : 라미닌 용액을 제거하고 파종 직전에 DPBS 두 번 코팅 된 표면을 씻어. </li><li> 계대 때 DMEM-F12에서 1 × 104 세포 / ㎠의 세포 현탁액 시드 (3 : 1) 상에 20 NG / ㎖ EGF 40 NG / ㎖의 bFGF로 보충 된 용지 (미리 예열 37 ° C까지) 라미닌은 배양 접시 코팅. </li><li> 37 ° C, 5 % CO 2 및&gt; 95 %의 습도에서 세포를 인큐베이션하고 세포를 3 일 동안 증식 할 수있다. </li><li> (1 3) 미디어가 37 ° C로 미리 예열, 10 ng의 신경 영양 인자 (BDNF) 파생 / ㎖ 뇌 보충 3 일 DMEM-F12를 포함하는 차별화 미디어와 미디어를 교체 한 후, 부착 된 신경 전구 세포를 구분합니다. </li><li> 분화 동안 3 일마다 미디어의 50 %를 교체합니다. 이십팔일 - 차별화는 약 21를 통해 점진적으로 발생합니다. 주의 : 세포 분화하기 시작하면, 하나의 TI의 매체의 50 %를 대체공기에 노출 같은 날 세포의 건강에 유해한 영향을 미칠 수있다. </li></ol>

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A. <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+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay.">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> J Vis Exp. (45), e2393 (2010).</li><li>Walker, T. L., Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+mouse,+two+cultures:+isolation+and+culture+of+adult+neural+stem+cells+from+the+two+neurogenic+zones+of+individual+mice.">One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice.</a> J Vis Exp. (84), e51225 (2014).</li><li>Lim, J. -. H., Koh, S., Olby, N. J., Piedrahita, J., Mariani, C. L. <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+characterization+of+neural+progenitor+cells+from+adult+canine+brains.">Isolation and characterization of neural progenitor cells from adult canine brains.</a> Am J Vet Res. 73 (12), 1963-1968 (2012).</li><li>Herranz, C., 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=Spontaneously+Arising+Canine+Glioma+as+a+Potential+Model+for+Human+Glioma.">Spontaneously Arising Canine Glioma as a Potential Model for Human Glioma.</a> J Comp Pathol. 154 (2), 169-179 (2016).</li><li>Walton, R. M., Parmentier, T., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neural+precursor+cell+regions+in+the+rostral+subventricular+zone,+hippocampal+subgranular+zone+and+cerebellum+of+the+dog+(Canis+lupus+familiaris).">Postnatal neural precursor cell regions in the rostral subventricular zone, hippocampal subgranular zone and cerebellum of the dog (Canis lupus familiaris).</a> Histochem Cell Biol. 139 (3), 415-429 (2013).</li><li>Walton, R. M., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+in+neural+progenitor+cells+in+a+dog+model+of+lysosomal+storage+disease.">Abnormalities in neural progenitor cells in a dog model of lysosomal storage disease.</a> J Neuropathol Exp Neurol. 66 (8), 760-769 (2007).</li><li>Milward, E. A., 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=Isolation+and+transplantation+of+multipotential+populations+of+epidermal+growth+factor-responsive,+neural+progenitor+cells+from+the+canine+brain.">Isolation and transplantation of multipotential populations of epidermal growth factor-responsive, neural progenitor cells from the canine brain.</a> J Neurosci Res. 50 (5), 862-871 (1997).</li><li>Ray, J., Peterson, D. A., Schinstine, M., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+differentiation,+and+long-term+culture+of+primary+hippocampal+neurons.">Proliferation, differentiation, and long-term culture of primary hippocampal neurons.</a> Proc. Natl. Acad. Sci. 90 (8), 3602-3606 (1993).</li><li>Palmer, T., Ray, J., Gage, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-2-responsive+neuronal+progenitors+reside+in+proliferative+and+quiescent+regions+of+the+adult+rodent+brain.">FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain.</a> Mol Cell Neurosci. 6 (5), 474-486 (1995).</li><li>Zhu, G., Mehler, M., Mabie, P., Kessler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+progenitor+cell+responsiveness+to+cytokines.">Developmental changes in progenitor cell responsiveness to cytokines.</a> J. Neurosci. Res. 56 (2), 131-145 (1999).</li><li>Whittemore, S. R., Morassutti, D. J., Walters, W. M., Liu, R. -. H., Magnuson, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen+and+substrate+differentially+affect+the+lineage+restriction+of+adult+rat+subventricular+zone+neural+precursor+cell+populations.">Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations.</a> Exp. Cell. Res. 252 (1), 75-95 (1999).</li><li>Kennedy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmortem+survival+characteristics+of+rat+glial+cells+in+culture.">Postmortem survival characteristics of rat glial cells in culture.</a> J Neurol Neurosurg Psychiatry. 50 (6), 798-800 (1987).</li><li>Viel, J. J., McManus, D. Q., Cady, C., Evans, M. S., Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+and+time+interval+for+culture+of+postmortem+neurons+from+adult+rat+cortex.">Temperature and time interval for culture of postmortem neurons from adult rat cortex.</a> J. Neurosci. Res. 64 (4), 311-321 (2001).</li><li>Huat, T. J., 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=IGF-1+enhances+cell+proliferation+and+survival+during+early+differentiation+of+mesenchymal+stem+cells+to+neural+progenitor-like+cells.">IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells.</a> BMC Neurosci. 15 (91), 1-13 (2014).</li><li>Supeno, N. 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=IGF-1+acts+as+controlling+switch+for+long-term+proliferation+and+maintenance+of+EGF/FGF-responsive+striatal+neural+stem+cells.">IGF-1 acts as controlling switch for long-term proliferation and maintenance of EGF/FGF-responsive striatal neural stem cells.</a> Int. J. Med. Sci. 10 (5), 522-531 (2013).</li><li>Ahmed, S., Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+enhances+the+differentiation+but+not+the+survival+of+CNS+stem+cell-derived+neuronal+precursors.">BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors.</a> J Neurosci. 15 (8), 5765-5778 (1995).</li><li>Wohl, C. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+enhances+neuronal+proliferation+and+astroglial+differentiation+in+cultures+of+CNS+stem+cell-derived+precursors.">Retinoic acid enhances neuronal proliferation and astroglial differentiation in cultures of CNS stem cell-derived precursors.</a> J Neurobiol. 37 (2), 281-290 (1998).</li><li>Araujo, D. M., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+effects+of+interleukin-4,-7+and-8+on+hippocampal+neuronal+cultures:+potential+involvement+of+glial-derived+factors.">Trophic effects of interleukin-4,-7 and-8 on hippocampal neuronal cultures: potential involvement of glial-derived factors.</a> Brain Res. 600 (1), 49-55 (1993).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Mol Neurobiol. 34 (3), 153-161 (2006).</li><li>Singec, I., 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=Defining+the+actual+sensitivity+and+specificity+of+the+neurosphere+assay+in+stem+cell+biology.">Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.</a> Nat Methods. 3 (10), 801-806 (2006).</li><li>Conti, L., 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=Niche-independent+symmetrical+self-renewal+of+a+mammalian+tissue+stem+cell.">Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.</a> PLoS Biol. 3 (9), 283 (2005).</li><li>Pollard, S. M., Conti, L., Sun, Y., Goffredo, D., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherent+neural+stem+(NS)+cells+from+fetal+and+adult+forebrain.">Adherent neural stem (NS) cells from fetal and adult forebrain.</a> Cereb Cortex. 16, 112-120 (2006).</li><li>Ma, W., 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=Cell-extracellular+matrix+interactions+regulate+neural+differentiation+of+human+embryonic+stem+cells.">Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells.</a> BMC Dev Biol. 8 (90), 1-13 (2008).</li><li>Goffredo, 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=Setting+the+conditions+for+efficient,+robust+and+reproducible+generation+of+functionally+active+neurons+from+adult+subventricular+zone-derived+neural+stem+cells.">Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells.</a> Cell Death Differ. 15 (12), 1847-1856 (2008).</li></ol>

The authors have nothing to disclose.

여기에 설명 된 프로토콜은 세포 생존 능력을 최대화하기위한 바람직한 배양 조건을 유지하기 위해 최적화된다. 추출, 분리 및 확장 동안 촬영 속도 및 관리가 매우 중요하다. 접착 단층 확장을 설정하기위한 중요한 단계는 기본 neurosphere를 효과적으로 분리한다. 통과 후, 충분히 neurosphere를 보조 부동 neurosphere를 생성 할 수 있습니다 해리. 미디어 변화하는 동안,이 neurosphere를이 제거 해리와 부착 ...

Regional variations in the rate of neurogenesis have been observed along the dorsoventral axis of the rodent hippocampus1,2. Furthermore, the rates of hippocampal neurogenesis also show distinct inter-species variation, with precursor cell turnover in the subgranular zone shown to be significantly lower in adult humans than in rodents3-5. Inter-species differences in hippocampal structural anatomy may be relevant here, as it has been postulated that neural stem cell distribution along the murine ventricular neuraxis may be influenced by cephalic flexures during embryological development6. To date, the rodent brain remains the most popular system in which to study adult neurogenesis. However, the brain of the domestic dog (Canis familiaris), with a size and structural organization intermediate between that of humans and rodents7, represents a valuable yet highly underused animal model. The canine hippocampus in particular embodies this structurally intermediate nature8-10 and can provide a unique perspective on intrinsic variations in neural precursor cell populations. With many closer parallels to the human brain, the canine model may also offer insight into the biology of adult human neurogenesis.
In vitro culture assays have become a key tool for the study of neural precursors and the cellular and biomolecular processes of adult neurogenesis. The neurosphere assay and adherent monolayer culture represent the two predominant systems for expanding neural precursor cells in vitro11-13. Protocols for brain extraction, hippocampal microdissection or neural precursor culture assays have been well documented for the rodent model14-16. However, for the adult canine brain they remain comparatively few17,18, focused instead on fetal or neonatal tissue19-21.
In our published study7 we investigated regional variations in neurogenesis and neural precursor cell populations across the dorsoventral axis of the adult canine hippocampus. Although highly dependent on breed, adulthood in canines is reached between 1 and 3 years of age. Here, we present detailed methods for the extraction, isolation and culture of neural precursor cells from the canine hippocampus. We provide systematic protocols for the expansion of neural precursor cells as both floating neurospheres and as an adherent monolayer culture, and for their subsequent differentiation into mature neural cell types.

<table><tbody><tr><td>1,000 &amp;mu;l filtered pipette tip</td><td>Axygen</td><td>TF1000</td><td></td></tr><tr><td>150 mm Petri dish</td><td>BD Biosciences</td><td>351058</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>Greiner Bio One</td><td>188271</td><td></td></tr><tr><td>200 &amp;mu;l filtered pipette tip</td><td>Axygen</td><td>TF200</td><td></td></tr><tr><td>24 well culture plate</td><td>Greiner Bio One</td><td>662160</td><td></td></tr><tr><td>35 mm tissue culture dish</td><td>BD Biosciences</td><td>353001</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>BD Biosciences</td><td>352340</td><td></td></tr><tr><td>6 well culture plate</td><td>BD Biosciences</td><td>351146</td><td></td></tr><tr><td>B-27 Supplement (50x) serum free</td><td>Life Technologies&amp;nbsp;</td><td>17504044</td><td></td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td>Life Technologies</td><td>13256029</td><td></td></tr><tr><td>Brain derived neurotrophic factor (BDNF)</td><td>Millipore</td><td>GF029</td><td></td></tr><tr><td>Collagen solution</td><td>Stem Cell Technologies</td><td>04902</td><td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&amp;nbsp;</td></tr><tr><td>DMEM (4.5 g/L, D-glucose) 500 ml</td><td>Life Technologies</td><td>11960044</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>Epidermal growth factor (EGF)</td><td>BD Biosciences</td><td>354001</td><td></td></tr><tr><td>F-12 nutrient mixture (Ham) (1x) Liquid</td><td>Life Technologies</td><td>31765035</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Life Technologies</td><td>16141079</td><td></td></tr><tr><td>Gelatin from Porcine Skin Type A</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>L-alanyl-L-glutamine dipeptide (GlutaMAX)</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Heparin sodium salt from (porcine)</td><td>Sigma-Aldrich</td><td>H314950KU</td><td></td></tr><tr><td>Laminin (mouse)</td><td>Life Technologies</td><td>23017015</td><td></td></tr><tr><td>NCFC serum free medium (NeuroCult)</td><td>Stem Cell Technologies</td><td>5720</td><td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&amp;nbsp;</td></tr><tr><td>Proliferation NS-A (NeuroCult)</td><td>Stem Cell Technologies</td><td>05773</td><td>Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771&amp;nbsp; from Stem Cell Technologies.</td></tr><tr><td>NSC basal medium (Rat; NeuroCult)</td><td>Stem Cell Technologies</td><td>5770</td><td>Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771&amp;nbsp;</td></tr><tr><td>Penicillin/Streptomycin (5,000 U/ml)</td><td>Life Technologies</td><td>15070063</td><td></td></tr><tr><td>Povidone-iodine</td><td>Munipharma</td><td>Betadine</td><td></td></tr><tr><td>Trypan blue (0.4%)</td><td>Life Technologies</td><td>15250061</td><td></td></tr><tr><td>Trypsin EDTA</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>Class II biological safety cabinet</td><td>ThermoFisher Scientific</td><td>Safe 2020 1.2</td><td></td></tr><tr><td>Brain knife (disposable)</td><td>Macroknife</td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td>ThermoFisher Scientific</td><td>HERAcell 150i</td><td></td></tr><tr><td>Centrifuge</td><td>Hettich</td><td>Universal 320R</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Dumont</td><td></td><td></td></tr><tr><td>Easypet Electric pipette</td><td>Eppindorf&amp;nbsp;</td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>Boeco</td><td>Bright-Line Improved Neubauer</td><td></td></tr><tr><td>Manual pipettes</td><td>Eppindorf research</td><td></td><td></td></tr><tr><td>Oscillating bone saw&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Scalpel blades (No.21)</td><td>Paramount</td><td></td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Delta</td><td></td><td></td></tr><tr><td>Water bath</td><td>Grant</td><td>JB Aqua 18 plus</td><td></td></tr></tbody></table>

isolation and expansion of adult canine hippocampal neural precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

시험 관내 신경 전구 분석법, 신경 및 신경 전구 세포 집단을 사용하여 특징 및 성인 송곳니 해마의 dorsoventral 축간에 비교 하였다. 절연 해마 조직 유래의 신경 전구 세포를 배양하여 100 μm의 28 일의 직경에 도달 분리 후 14 일 이내에 플로팅 neurosphere를 형성. 지느러미와 복부 균주에서 유래 neurosphere를가 평균 크기에 차이가 없었다, 단일 세포로 효소 분해 다음, neuro...

Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

송곳니 뇌는 성인 신경 연구에 귀중한 모델입니다. 분리 및 기본 뇌 조직에서 성인 개 해마 신경 전구 세포 확장을위한 프로토콜은 여기에 표시됩니다.

분리 및 성인 개 해마 신경 전구체의 확장

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

isolation-and-expansion-of-adult-canine-hippocampal-neural-precursors

Research

JoVE Journal

Developmental Biology

7.0K 조회수.  University of Sydney.  송곳니 뇌는 성인 신경 연구에 귀중한 모델입니다. 분리 및 기본 뇌 조직에서 성인 개 해마 신경 전구 세포 확장을위한 프로토콜은 여기에 표시됩니다. 

비디오: Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

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 animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

성인 Hippocampal 북극 그라운드 다람쥐 신경 줄기 세포의 성장과 분화

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

연구

신경과학

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

절연 및 Neurosphere 어세를 사용하여 성인 마우스 신경 줄기 세포의 확장

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

쥐의 해마 영역의 치아 이랑 (dentate gyrus)에서 수지상 Arborization의 평가

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

동일한 호스트 종에서 항체와 면역 조직 화학 및 다중 라벨은 성인 해마 신경 발생을 연구하기 위해

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

셀 성인 마우스 뇌실 영역에서 신경 줄기 및 전구 세포의 정렬과 세포주기 역학의 라이브 이미징

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

마우스 뇌량 밑 영역에서의 분리 및 성인 신경 줄기 세포의 문화

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

성인 해마 신경 전구세포의 회로 특정 조절 을 시술

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

산후 (P1−3) 마우스 신경성 틈새에서 신경구의 격리 그리고 확장

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

<meta charset="utf8"></head><body>쥐 뇌실하 영역에서 분리된 NSC의 효율적인 핵 분석

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>저자 스포트라이트: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...