우리는 비디오 제작 및 편집, 데이터 수집에 도움 매트 쿡의 전문 기술 지원 에반 다이사트 및 마크 레너드를 감사하고, 전문 편집 지원 사라 Gelbard. 작품은 SALB, SALB 및 SF에 건강 연구 프로그램 부여에서 전략적 교육 이니셔티브 (TGF Neurodegenerative Lipidomics에서 CIHR 교육 프로그램, 9121), 그리고 인프라 건강 연구의 캐나다 연구소 (CIHR, 청소 62,626)에서 운영 기금에 의해 투자되었다 캐나다 온타리오 재단 혁신과 혁신 트러스트에서 SF로 지원합니다. SI는 파킨슨 연구 컨소시엄의 공헌과 과학 기술의 온타리오 대학원 장학금을 받았다. NV는 노화와 이후 전문적인 교제 Neurodegenerative Lipidomics에서 CIHR 교육 프로그램의 연구소를받습니다. 우리는 기꺼이 오토 데스크 리서치에서 제공하는 교육 소프트웨어 및 기술 지원을 인정합니다.

<ol>
	<li>Kempermann, G., Jessberger, S., Steiner, B., Kronenberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milestones+of+neuronal+development+in+the+adult+hippocampus.">Milestones of neuronal development in the adult hippocampus.</a> Trends Neurosci. 27, 447-452 (2004).</li><li>Campos, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurospheres:+insights+into+neural+stem+cell+biology.">Neurospheres: insights into neural stem cell biology.</a> J Neurosci Res. 78, 761-769 (2004).</li><li>Imbeault, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+controls+gap+junction+protein+expression+and+function+in+postnatal+hippocampal+neural+progenitor+cells.">The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells.</a> BMC Neurosci. 10, 13-13 (2009).</li></ol>

동물의 모든 실험은 오타와 애니멀 케어위원회의 대학과 동물 케어의 캐나다 협의회가 정한 지침과 규정에 따라 수행되었다.

이 프로토콜은 문화와 순차적으로 cryosection 이후 나탈 hippocampal NPCs에게 절차를 설명하는 것은 면역에 의해 단백질 표현을 집중하고, 마지막으로 전체 3D neurosphere 이내 immunopositive 세포의 지형 위치를 재구성하고 분석합니다. 세포 생물학 문화와 가공 기술, 현미경 이미징을 (OpenLab, 순발력 및 기타 이미지 분석 소프트웨어) 결합하여, 그래픽 편집 소프트웨어 용량 (어도비 포토샵) 및 3D 애니메이션 및 합성 소프트웨어 용량 (오토 데스크 마야)는, 우리는 재구 성할 수있는 방법론을 제시 neurosphere 문화 전반에 걸쳐 휴대폰 위치와 단백질 표현의 충실 재건에 대한 수 있도록 일련 2D 이미지에서 3D 문화의 모든 세포 구성. 이 프로토콜은 등록과 두꺼운 시리얼 섹션을 통해 epifluorescent 얇은 시리얼 섹션 또는 공촛점 Z - 스택의 재건과 동일 효과와 함께하실 수 있습니다. 포스트 나탈 머리에 neurogenesis 및 gliogenesis 과정을 통해 관찰 단계의 많은 neurosphere 문화 recapitulates 단일 NPCs의 clonal 확장 때문에, 그 3 차원 구조의 영향은 동일한 실험 환경 1 노출 자손에 중요한 NPC 운명의 결정자를 나타냅니다. 시리얼 neurosphere 섹션의 핵심과 주변에서 혈통과 단백질 표현의 발산은 세포 위치, 기계적 응력과 전단 강제를 포함하여 여러 물리적인 요소가 NPC 생물학 2 조절에서 중요한 역할을 제안합니다. 또한, connexin 단백질 표현, 여기에 분석은 NPCs가 3D 정지 neurospheres 또는 세포 플라스틱 기판이나 3D 무료 양식 여부 변경 기능 connexin - 중재 통신 laminin 기판에 도금으로 양식하는 경우에 따라 변경 표시되었습니다 - 부동 문화 3. NPC의 운명에 대한 세포 위치의 영향은 불분명 남아 있습니다. 그것은 기술적으로 혈통 및 신호 단백질의 antigenic 평가는 3D 건축의 중단은 세포 permeabilization 모든 세포에 항체 액세스를 활성화하는 데 필요한 특정 NPC 생물학에 3D 문화 이내 공간적 위치의 영향을 분석하는 도전이다. 여기, 우리가 하이브리드 시각화 방법론 특정 단백질이 3D 문화의 독특한 위치 localizations 전시 여부를 결정하는 수단을 제공한다는 것을 보여주기, 가장 중요한 아마도 neurosphere 이내 지역 현지화 관련하여 단백질 기능과 규제를 추론하고 테스트하는 데 사용하고, 수 , 3D 문화의 NPC 운명에 3D 지형 및 셀룰러 위치의 영향을 연구하는 데 필요한 위치 데이터를 제공합니다.

프로토콜 목록에 * 다른 모든 표준 화학 시약은 시그마 - 알드리치에서 있습니다. 다른 모든 표준 조직 문화 시약는 Invitrogen에서 있습니다.

localizing protein in 3d neural stem cell culture: a hybrid visualization methodology

신경 줄기 및 전구 세포에 영향을 미치는 3 차원 (3D) 지형의 중요성 (NPC) 표현형 널리 아직 공부 도전 인정받고 있습니다. 배아 또는 사후 나탈 머리에서하면 dissociated, 단일 NPCs는 neurospheres을 형성 중지 분아 따위에 의해 번식합니다. 이러한 문화 이내 딸 세포가 저절로 같은 세포외 환경에 노출 임에도 불구하고 확장의 과정을 통해 서로 다른 발달 lineages을 (뉴런, oligodendrocytes 및 astrocytes) 채택. 이 진행의 recapitulates이 단계의 대부분은 이후 나탈 두뇌에서 neurogenesis 및 gliogenesis 과정을 통해 관찰하고 종종 통제 환경에서 기본적인 NPC의 생물학을 연구하는 데 사용됩니다. NPC의 운명에서 이러한 문화 이내 3D 지형 및 셀룰러 위치의 전체 영향을 평가하는 것은, 그러나, 어렵습니다. 대상 단백질을 집중하고 immunocytochemistry하여 NPC의 lineages를 확인하려면 자유 떠있 neurospheres은 기판에 도금 또는 순차적으로 sectioned해야합니다. 이 처리는 동일한 세포 permeabilization과 영역에 걸쳐 항체 접근을 보장하기 위해 필요합니다. 그 결과, cryosections 또는 3D Z - 스택의 공촛점 reconstructions의 2D epifluorescent 이미지는 별도의 물리적 또는 디지털 3D 조각 내의 세포의 위치에 대한 공간 정보를 제공할 수 있고 손상 영역에 휴대폰 위치를 시각화하지 않습니다. 여기 neurosphere 문화의 지형을 강조하고 전체 문화 전반에 걸쳐 단백질 표현의 공간적 분석을 허용하는, 우리는 고립을위한 프로토콜, 확장하고, 대상 단백질의 epifluorescent 또는 공촛점 면역에 적합한 사후 나탈 hippocampal neurospheres 일련 sectioning을 제시. Connexin29 (Cx29)을 예로 분석이다. 다음 그래픽 편집 및 3D 모델링의 하이브리드를 사용하는 것은 엄격하게 생물 학적 세부 사항을 유지하기 위해 적용되는 소프트웨어, 우리는 이러한 이미지의 3D 구조 위치를 다시 조립하고 디지털 전체 neurosphere 내에 표시된 세포를지도하는 방법에 대해 설명합니다. 이 방법론은 대상 단백질 및 전체 3D 문화 지형을 통해 세포의 세포 위치 시각화 및 분석을 가능하게하고 체외에서 neurogenesis 및 gliogenesis 과정을 통해 세포 사이의 공간 관계의 상세한 분석을 용이하게합니다. 임보와 발렌 주 엘라 모두 동등하게 기여 공동 첫번째 작가로 간주되어야합니다.

Watch this Scientific Journal Video about Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology at JoVE.com

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

여기, 우리는 세 차원 (3D) 문화, 생산 확장하고 immunolabel 출생 후의 hippocampal 신경 전구 세포 (NPCs)하는 방법에 대해 설명합니다. 다음, 하이브리드 시각화 기술을 사용, 우리는 immunolabelled cryosections의 디지털 이미지가 전체 차원 neurosphere에 걸쳐 immunopositive 세포의 공간적 위치를 재구성하고지도하는 데 사용할 수있는 방법을 보여줍니다.

3D 신경 줄기 세포 문화 번역하는 단백질 : 하이브리드 시각화 방법론

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

localizing-protein-3d-neural-stem-cell-culture-hybrid-visualization

Research

JoVE Journal

Neuroscience

12.6K 조회수.  University of Ottawa. 여기, 우리는 세 차원 (3D) 문화, 생산 확장하고 immunolabel 출생 후의 hippocampal 신경 전구 세포 (NPCs)하는 방법에 대해 설명합니다. 다음, 하이브리드 시각화 기술을 사용, 우리는 immunolabelled cryosections의 디지털 이미지가 전체 차원 neurosphere에 걸쳐 immunopositive 세포의 공간적 위치를 재구성하고지도하는 데 사용할 수있는 방법을 보여줍니다.

비디오: Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

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 of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

배아 척수에서 뉴런의 Commissural 해부와 문화

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

연구

신경과학

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 ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Neurosphere 분석을 사용하여 마우스 배아 신경 줄기 세포 문화 구축

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

세포 어세를 형성 신경 콜로니 : 신경 전구 세포로부터 타고난 신경 줄기 세포를 차별 어세이

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neuroblast 분석 : 유동세포계측법을 사용하여 차별화 신경 줄기 세포에서 자손의 연결 전구 세포의 생성과 강화에 대한 분석

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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 ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

신경 줄기 세포의 열거 클론 세이를 이용하여

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

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 ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

성인 인간 피부 섬유 아 세포에서 유도 된 신경의 고수익 문화의 간단한 세대

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

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.