이 작품은 NIH 보조금 R01AR054593와 슈에 R01AR054593-S1에 의해 지원됩니다.

1. 조직 문화 플레이트의 작성 <ol><li> 코트는 각이 잘 폴리-D-리신 (PDL)이 우물의 바닥을 커버 할 수있을만큼. 플레이트는 후드에서 건조하도록 허용합니다. </li><li> 우물이 건조 후, 멸균 물을 헹구고, 대기음. 플레이트는 후드에서 건조하도록 허용합니다. </li><li> 때 fibronectin과 건조, 코트 (6 ML 1 밀리그램의 농도에서 37 ° C에서 하룻밤 biowhittaker 물에 용해 된 것). </li><li> NCSC 매체를 추가합니다 [95 ML DMEM/F12, 1 ML 펜 / 패 혈성 (P / S), 1 ML N2, 2 ML B27, 100 μl 메르 캅토 에탄올 (2ME, 50 MM 주식), bFGF는 (20 NG / ML 중간), IGF -1 (20 NG / 매체의 ML)과 EGF (20 NG / 매체의 ML)] 접시에 건조하기 전에 fibronectin, </li></ol> 2. 인간의 두피에서 헤어 소낭 세포를 추출 <ol><li> 분리 NCSCs의 절차를 시작하기 전에, 하나는 (표 1 참조) 각 매체와 시약을 준비해야합니다. </li><li> 새롭게 단장 절차 나 FE 신선한 성인의 두피 피부할거야, 두피 조직은 PBS의 페니실린 - 스트렙토 마이신을 포함하는으로 씻어 수집하고 있습니다. </li><li> 피부 후 50 ML 튜브로 옮겨와 4 ° C.에서 하루 아침에 Dispase (10 MG / ML)과 DMEM에 incubated 수 있습니다 37 번 2-4 시간을위한 부화는 ° C도 효과적입니다. 피부 조각이 관통하는 효소를 허용하는 1cm의 최대 너비해야합니다. </li><li> 소독 페트리 접시에 피부를 전송하고, 피부 표면 근처에 머리 샤프트​​을 쥐었고 단단히하고 부드럽게 당겨 피부의 각 머리를 당긴다. 모낭은 anagen (그림 1A) 또는 telogen (그림 1B) 또는의 형태를 보여줍니다. </li><li> 주기적으로 진동과 실내 온도 15-20 분 동안 0.05 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) (Invitrogen)에 절연 고치 조각을 길러, 반응을 중지 10 % FBS와 4 ML DMEM을 추가합니다. follicular 상피는 trypsinized와 다양한 크기와 모양의 세포를 포함하는 단일 셀 정지를 얻기 위해 필터 40 μm를 통해 필터링됩니다. </li><li> 회전5 분 200 XG에, 1 ML PBS는 2 % 혈청 (FBS)을 포함에 다시 중지 조심스럽게 표면에 뜨는 폐기하십시오. </li><li> 또한 뽑혀 모낭은 원위치에 헤어 분야를 성장 트립신 소화하지 않고 문화에 배치 할 수 있습니다. </li></ol> 3. 흐름 Cytometric 셀 정렬을 사용하여 헤어 소낭의 NCSCs의 절연 <ol><li> 헤어 대과 단일 셀 정지는 트립신의 소화에 의해 획득 및 CD271 (APC-복합) / HNK1 (FITC-복합) 또는 CD271 / alpha4 어둠 속에서 얼음에 40 분 인테그린 (PE-복합)에 대한 항체로 분류되었습니다. </li><li> 상온에서 200 XG에서 5 분 동안 원심 분리기와 표면에 뜨는을 대기음. </li><li> PBS는 2 % 혈청 (FBS)을 포함에 Resuspend 세포 및 정렬하기 전에 PI는 죽은 세포 밖으로 게이트에 추가됩니다. </li><li> 유동 세포 계측법 (FACS)에 의해 세포 정렬을 수행합니다. CD271를 수집 +, HNK1 + 더블 긍정적 인 셀 또는 CD271 +, alpha4 인테그린 + 더블 긍정적 인 세포 (그림 2). </li><li> 후 정렬ING, CD271 +, HNK1 + 더블 긍정적 인 셀 또는 CD271 +, alpha4 인테그린 + 더블 긍정적 인 셀은 NCSC 매체에 초저 첨부 파일 접시에 배양 아르 </li></ol> 4. 기본 NCSCs의 문화 <ol><li> 문화 disassociated follicular 세포 또는 FACS는 NCSC 매체의 초저 첨부 파일 접시에 세포를 정렬 매일 매체를 변경합니다. </li><li> 현미경으로 매일 세포 배양을 확인합니다. NCSCs는 여러 일 기증자 (그림 3A)의 나이에 따라 문화에 2~5주에서 잘 구성된 영역 이후 부동 작은 응집체를 형성하기 시작합니다. 가지가 솟아 오른 지역 여러 주 동안 현장에서 잘 구성된 영역 (그림 3B) 전체 모낭은 NCSC 매체에 배양 아르에서 몇 일에 나타납니다. </li></ol> 5. NCSCs의 확장 <ol><li> 한 번 PBS로 세포를 씻으십시오. </li><li> 사전 예열을 추가 (37 ° C, 임계) 37시 accutase 및 배양 ° C 5-10에하나의 셀에 있는지 신경 줄기 세포 분야는 dissociated하기 위해 주기적으로 진동이있는 분 (첨부 파일 양식 NCSC, 37에서 accutase과 배양 세포에 대한 확인 헤어 소낭의 줄기 세포 분리하기 위해 5 분에 대한 ° C). </li><li> 남아있는 accutase 솔루션을 제거 할 DMEM/F12 중간에 상온 200 XG에서 5 분에 두 번 세척하고 세포를 원심 분리기. </li><li> NCSC 문화 매체와 세포를 다시 일시 중지합니다. </li><li> 부동 문화를 들어, 초저 첨부 파일 플레이트에 세포를 넣어. 첨부 파일 문화를 들어, pretreated 조직 배양 플레이트에 세포를 넣어. </li></ol> 6. 대표 결과 NCSC 문화 매체 피더 셀 필요없이 undifferentiated 상태에서 hNCSCs를 유지하기에 충분합니다. Keratinocytes는 세포 분열 따위에 의해 번식하고 점차 매체에서 죽었되지 않습니다. 특정 작고 둥근 세포가 세포 분열 따위에 의해 번식하고 3-5일 후 정지의 작은 집합체를 형성합니다. 이러한 부동 집계는 천천히 증가우리가 헤어 분야 (그림 3A)이라고한다 N의 크기, 생성 입체 영역과 같은 구조. 코팅 접시에 배양하면 NCSCs는 표면에 부착하고 영역을 형성하지 않습니다. 첨부 NCSCs는 중지보다 더 빠르게 성장합니다. 영역 - 성형 또는 첨부 된 줄기 세포는 NCSCs 마커를 표현한다. 전체 모낭이 문화가되면 영역이 솟아 오른 지역 (그림 3B)에 해당하는 지역에서 형성된다. <img alt="그림 1" src="/files/ftp_upload/3194/3194fig1.jpg" /> 1 그림. anagen (A)와 telogen (B) 모낭을 뜯어 낸. <img alt="그림 2" src="/files/ftp_upload/3194/3194fig2.jpg" /> 그림 2. NCSCs의 FACS 분석의 대표 이미지 왼쪽 패널 :. 셀 안티 CD271 및 안티 alphe 4 인테그린 항체를 사용하여 출입을 금지합니다. 오른쪽 패널 : 셀 안티 CD271 및 안티 HNK1 항체를 사용하여 출입을 금지 아르. <img alt="그림 3" src="/files/ftp_upload/3194/3194fig3.jpg" /> 그림 3. 헤어 분야의 형태 부동 헤어 분야, 왼쪽 패널의 (A) 형태론 :. 저전력, 오른쪽 패널 :. 높은 전력 (B) 솟아 오른 지역, 왼쪽 패널에서 원래의 장소에 헤어 영역의 형태론 : telogen의 팽창에서 헤어 영역, 오른쪽 패널 : anagen 헤어 소낭의 볼록한 지역에서 헤어 영역.

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우리는 공개 할 아무 것도 없습니다.

설명 셀 절연과 문화 방법은 재현하고 강력한 있습니다. 우리는 폭 넓은 연령대에 걸쳐 개인의 수십에서 NCSCs를 생성했습니다. 그것은 바로 조직의 수확 후 조직을 처리하는 것이 좋습니다 있지만, 우리는 두피의 조직이 안전하게 세포 생존에 미치는 영향을 최소화 하루 아침에 교통 얼음의 미디어에 저장할 수 있습니다 것으로 나타났습니다. 이 항생제로 두피 조직을 치료?...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>11965-092</td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>Invitrogen</td>
 <td>11330-32</td>
 </tr>
 <tr>
 <td>Heat-inactivated FBS</td>
 <td>Hyclone</td>
 <td>SH30071.03</td>
 </tr>
 <tr>
 <td>B27 supplement</td>
 <td>Invitrogen</td>
 <td>17504044</td>
 </tr>
 <tr>
 <td>N2 supplement</td>
 <td>Invitrogen</td>
 <td>17502048</td>
 </tr>
 <tr>
 <td>bFGF</td>
 <td>Invitrogen</td>
 <td>PHG0026</td>
 </tr>
 <tr>
 <td>EGF</td>
 <td>R&amp;D system</td>
 <td>236-EG-01M</td>
 </tr>
 <tr>
 <td>IGF-I</td>
 <td>R&amp;D system</td>
 <td>291-G1-050</td>
 </tr>
 <tr>
 <td>0.05% Trypsin/EDTA</td>
 <td>Invitrogen</td>
 <td>25300-054</td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>Invitrogen</td>
 <td>17105041</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15070063</td>
 </tr>
 </tbody> 
</table>
Table 1.

isolation and culture of neural crest stem cells from human hair follicles

모낭은 평생 성장을 받아야과 헤어 사이클은 줄기 세포 증식 및 정지를 포함 잘 제어 과정입니다. 헤어 팽창은 성인의 줄기 세포 1에 대한 잘 특징 틈새 시장입니다. 바깥 쪽 루트 피복의이 부분은 ​​상피 줄기 세포 2, melanocyte 줄기 세포 3 4-7 줄기 세포와 같은 신경 문장을 포함한 줄기 세포의 다른 유형의 번호를 포함하고 있습니다. 모낭은 인간 줄기 세포의 다른 유형에 대한 접근과 풍부한 소스를 나타냅니다. 우리와 다른 사람들은 4,5 인간의 태아 및 성인 모낭에서 신경 문장의 줄기 세포를 (NCSCs) 분리했습니다. 이러한 인간의 줄기 세포는 라벨 - 유지 전지이며, 체외에서 비대칭 세포 분열을 통해 자기 갱신 할 수 있습니다. 그들은이지나 신경 크레스트 세포 마커가 아닌 차별화 마커를 표현한다. 우리 식 프로파일 연구들은 손쥐 피부에 미숙 한 신경 cre와 유사한 유전자 발현 패턴을 공유했다성 세포. 그들은 체외 clonal 하나의 세포 배양에 후, myogenic melanocytic, 그리고 neuronal 세포 lineages을 야기 할 수 있습니다 clonal multipotency을 나타냅니다. 차별화 된 세포는 계보 (Lineage) - 특정 마커를 취득뿐만 아니라 전 생체 조건에 적합한 기능을 보여줍니다 없습니다. 또한 이러한 NCSCs는 중간 엽 lineages 대한 차별화의 잠재력을 보여줍니다. 차별화 된 neuronal 세포는 마우스의 뇌에 계속하고 neuronal 차별화 마커를 유지 할 수 있습니다. 그것은 머리가 NCSCs 신경 regrowth을 할 수 있습니다 파생 고치, 그리고 그들이 끊어 버렸네 척추 부상 8 다음이 줄기 세포를 이식 마우스에 모터 기능을 향상시키는 것이 보여왔다. 또한, 주변 신경은 줄기 세포 이식은 9, 눌린 좌골 신경에 인접 해 피부 파생 전구체 세포의 주입과 수리 된 remyelination 10 결과입니다. 따라서 헤어 대과 / 피부 파생 NCSCs은 이미 연구에 대한 긍정적 인 결과를 보여 주었다잠복기 모델 egenerative 치료. 유도 만능 줄기 (IPS) 셀에 재 프로그램 체세포는 재생 의료에 대한 엄청난 잠재력을 보여 주었다. 그러나, 특히 IPS 세포, 종양 유전자 / 바이러스 통합 만능 줄기 세포의 잠재적 tumorigenicity의 장기적 효과 아직 많은 문제를 적절하게 해결되지 않은이 있습니다. IPS 세포는 재생 의료에 사용하기 전에 극복 할 많은 장애물은 여전히​​ 있습니다. 성인 줄기 세포는 안전한 것으로 알려져 있으며, 그들은 같은 골수 이식 등 많은 년에 임상 적으로 사용되었습니다 반면. 대부분의 환자는 이미 치료의 혜택을했습니다. Autologous 성인 줄기 세포는 아직 이식에 대한 선호 세포입니다. 따라서, 인간의 피부 / 머리카락 모낭에 쉽게 액세스 할 수 있으며 확장이 성인 줄기 세포는 재생 의료에 대한 가치있는 소스입니다.

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles at JoVE.com

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

이 문서는 인간의 모낭에서 신경 문장의 줄기 세포의 분리와 문화에 대한 강력한 프로토콜을 제공합니다.

인간의 모공에서 신경 크레스트 줄기 세포의 분리와 문화

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

isolation-culture-neural-crest-stem-cells-from-human-hair

Research

JoVE Journal

Neuroscience

16.8K 조회수.  School of Medicine, University of Pennsylvania. 이 문서는 인간의 모낭에서 신경 문장의 줄기 세포의 분리와 문화에 대한 강력한 프로토콜을 제공합니다.

비디오: Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

Murine 신경 크레스트 줄기 세포의 배양 칙의 배아 추출 생산

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

연구

신경과학

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

폐기 인간 태아 두피 조직에서 신경 줄기 세포의 생성

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

쥐 배아 신경 세포의 분리 및 문화 : 빠른 프로토콜

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine 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 mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

배아 Murine 신경 튜브에서 신경 크레스트 세포의 분리 및 문화

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

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

하나의 마우스, 두 문화 : 분리 및 성인 신경의 문화가 개인 마우스의 두 신경 인성 영역에서 줄기 세포

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

인간 다 능성에서 신경 능선 세포의 전구 세포의 공급이없는 유도는 줄기 세포

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

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 ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

쥐의 눈으로 인간 망막 안료 상피 세포의 트랜스 scleral Subretinal 이식에 대 한 세련 된 프로토콜의 개발

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

절연 및 마우스 배아 신경 줄기 세포의 문화

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

인간 포피로부터의 일차 슈완 세포, 각질세포 및 섬유아세포의 분리, 배양 및 특성화

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...