Biz eleştiri ve bu raporun düzenleme için Dr Soshana Svendsen teşekkür ederim. Bu çalışma NIH / NINDS 1U24NS078370-01 ve CIRM DR2A-05320 ile katkıda bulunmuştur.

Yazarlar ifşa hiçbir şey yok.

<img alt="Şekil 6," fo:content-width="5in" src="/files/ftp_upload/51219/51219fig6highres.jpg" width="500" /> Şekil 6.. Doğrama şematik. Mekanik kesme yöntemini kullanarak kültür içinde sfero kök / progenitör hücreler genişletilmesi. Kritik Adımlar Kıyma genişleme paradigmasının bir bakış. HNPC küre boyutu neurospheres pasaj önce gözlemlemek için önemli kriterlerden biridir <strong...

A correction was made to <a href="/video/51219">A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue</a>. The corresponding author was changed from:
Brandon C. Shelley 
to:
Clive Svendsen

A correction was made to <a href="/video/51219">A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue</a>. The corresponding author was changed.

Erratum: A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

Bir tek tabaka 1-3 veya toplam neurospheres 4-7 ya gibi kültür kemirgen sinir kök hücreleri genişleyen uzun bir geçmişi vardır. Buna ek olarak, gelişmekte olan merkezi sinir sisteminin 8-17 çeşitli bölgelerinden izole insan nöral progenitör hücrelerin (hNPCs) in vitro genişletilmiştir. Bu hücreler iki güçlü, astrositlerde ve nöronlar hem de farklılaşma yeteneğine sahiptirler ve nöral gelişme 18,19 ve hastalık mekanizması 20,21 okuyan çok yararlı bir araç olmuştur. hNPCs ayrıca entegrasyon, hayatta kalma ve fonksiyonel etkileri 22-24 çeşitli düzeylerde ile santral sinir sistemi hastalığı birçok farklı hayvan modellerinde naklediliyor edilmiştir. Genellikle epidermal büyüme faktörü (EGF) ve / veya fibroblast büyüme faktörü-2 (FGF-2), 25-28 - - ve yapışkan 29 ve hem de üç Geleneksel olarak, kemirgen ya da insan fetal NPC-türevi büyüme faktörleri maruzBoyutlu küremsi sistemleri tipik olarak bir tek hücre süspansiyonu halinde 30-34 enzimatik ayrılma ile pasajlanır. Araştırma ya da klinik kullanım için hücreleri genişletmek için bir standart yöntem kolay manipülasyon nedeniyle yapışkan bir tek tabaka olduğu gibi. Bununla birlikte, enzimatik veya kimyasal çözeltiler ile pasajı tek tabaka ve neurosphere hNPCs erken yaşlanma 35 ile sonuçlandığını göstermiştir. Buna ek olarak, enzimatik ayrışma embriyonik kök hücreler 36-38 ile gösterilen verilere dayalı olarak farklılaşma ve kromozomlarda anormallikleri artan seviyeleri ile sonuçlanabilir. Pasajlanması hNPCs standart yöntem mevcut iyi imalat uygulamaları fazına 1 klinik çalışmalarda (Hücreler Inc, Neuralstem Inc Stem) gitti (cGMP) sınıf ürünler üretti rağmen, yöntem sınırlayıcı, hücre büyütme sadece birkaç mermi izin potansiyeli bankacılık. Açıkçası, büyük araştırma deneyleri ve gelecekteki klinik denemeleri yeteneği yararlanabilecekbüyük ölçekli büyüme ve hücre bankacılığı izin toplu olarak ve gecikmeli yaşlanması ile hücreleri yaymak. Bu ihtiyacı karşılamak için, hücre-hücre teması korumak için küçük kümeler halinde onları &quot;kesme&quot; bir roman ve mekanik Pasajlanması bozulmamış neurospheres otomatik bir yol geliştirdi. Bu yöntem büyük bir alternatif 3 boyutlu biyoreaktör kültürü yöntemi 40 ile görüldüğü gibi bunların ömrü 39 ve süspansiyon kültürü, tek tabakalı kültürler ile karşılaştırıldığında kuluçka alanı daha verimli kullanımına izin verir artmıştır. Resim kıyma protokol geçişi 10, standart yöntemler kullanılarak bir pasajı olası bir feat daha büyük bir fetal numuneden büyük ölçekli bankaların üretimine olanak sağlamaktadır. Pasajı hNPCs için bu yöntem alışılmamış iken, popülaritesi artmaktadır ve yakın zamanda, bu tür insan embriyonik ve uyarılmış pluripotent kök hücrelerinden elde edilen nöral kök hücreleri gibi diğer hücre tiplerinde, v dahil olmak üzere çeşitli uygulamalar için geniş ölçekli bir genişleme sağlayan yayınlanmıştıritro hastalık modelleme 41-46. Daha da önemlisi, bir cGMP dereceli hNPC hücre bankası zaten tekniğin gelecekteki klinik uygulamalar karşı uygulanabilir olduğunu gösteren, kesme yöntemi ile üretilmiştir.

<table><tbody><tr><td>Beaker, 50 ml</td><td>Fisherbrand</td><td>FB-100-50</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Bio-Safety Cabinet, class II</td><td>Baker</td><td>SG-603A</td><td>4 ft. or 6 ft. model.&amp;nbsp; 6 ft. model recommended; multiple manufacturers/suppliers</td></tr><tr><td>Blades, Double-edge Prep</td><td>Personna</td><td>74-0002</td><td>multiple manufacturers/suppliers.&amp;nbsp; CAUTION: Sharp</td></tr><tr><td>Cell Freezing Media</td><td>Sigma-Aldrich</td><td>C6295-50ML</td><td>DMSO, serum-free</td></tr><tr><td>Centrifuge, swing-bucket with 15 ml inserts</td><td>Eppendorf</td><td>5810 R</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Conical Tubes, 15 ml</td><td>Fisherbrand</td><td>S50712</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Conical Tubes, 50 ml</td><td>BD Falcon</td><td>352074</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Controlled Rate Freezer</td><td>Planer</td><td>Kryo 750</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Cryovials, 2 ml</td><td>Corning</td><td>430488</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Culture Flask, Vented, T12.5</td><td>BD Falcon</td><td>353107</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Culture Flask, Vented, T25</td><td>BD Falcon</td><td>353081</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Culture Flask, Vented, T175</td><td>BD Falcon</td><td>353045</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Culture Flask, Vented, T75&amp;nbsp;</td><td>BD Falcon</td><td>353110</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Filter, 0.22 &amp;micro;m, attached cup, 1 L</td><td>Millipore</td><td>SCGPU11RE</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Filter, 0.22 &amp;micro;m, attached cup, 150 ml</td><td>Millipore</td><td>SCGVU01RE</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Filter, 0.22 &amp;micro;m, attached cup, 500 ml</td><td>Millipore</td><td>SCGPU05RE</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Filter, 0.22 &amp;micro;m, attached cup, 50 ml</td><td>Millipore</td><td>SCGP00525</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Filter Paper, 8.5 cm circles</td><td>Whatman/GE</td><td>1001-085</td><td></td></tr><tr><td>Forceps, Standard Pattern - Serrated/Curved/18 cm</td><td>Fine Science Tools</td><td>11001-18</td><td></td></tr><tr><td>Freezing Chamber, Isopropyl Alcohol</td><td>Nalgene</td><td>5100-0001</td><td>&quot;Mr. Frosty&quot;</td></tr><tr><td>Incubator, 37 &amp;deg;C/5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Forma</td><td>370 series</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Hemacytometer, Phase</td><td>Hausser Scientific</td><td>1475</td><td>multiple manufacturers/suppliers</td></tr><tr><td>McIlwain Tissue Chopper</td><td>Lafayette Instruments</td><td>TC752-PD</td><td>&amp;nbsp;Petri dish modification required.&amp;nbsp;&amp;nbsp; CAUTION:&amp;nbsp; Moving, sharp blade.</td></tr><tr><td>Micropipettor, 1-10 &amp;mu;l</td><td>Gilson</td><td>F144562</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Micropipettor, 100-1,000 &amp;mu;l (starter kit)</td><td>Gilson</td><td>F167700</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Micropipettor, 2-20 &amp;mu;l (starter kit)</td><td>Gilson</td><td>F167700</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Micropipettor, 20-200 &amp;mu;l (starter kit)</td><td>Gilson</td><td>F167700</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Nutdriver, Autoclavable, 5/16&quot;</td><td>Steritool</td><td>10302</td><td></td></tr><tr><td>Pasteur Pipets, cotton-plugged</td><td>Fisherbrand</td><td>13-678-8B</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Petri Dish, Glass, Autoclavable</td><td>Corning</td><td>3160-100</td><td></td></tr><tr><td>Pipet Aid</td><td>Drummond</td><td>4-000-101</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Shim disc</td><td>McMaster-Carr</td><td>VARIABLE</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile barrier pipet tips, 10 &amp;mu;l</td><td>AvantGuard</td><td>AV10R-H</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile barrier pipet tips, 1,000 &amp;mu;l</td><td>AvantGuard</td><td>AV1000</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile barrier pipet tips, 20 &amp;mu;l</td><td>AvantGuard</td><td>AV20-H</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile barrier pipet tips, 200 &amp;mu;l</td><td>AvantGuard</td><td>AV200-H</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile Disposable pipettes, all-plastic wrap, 10 ml</td><td>Fisherbrand</td><td>13-676-10J</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile Disposable pipettes, all-plastic wrap, 2 ml</td><td>Fisherbrand</td><td>13-675-3C</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile Disposable pipettes, all-plastic wrap, 25 ml</td><td>Fisherbrand</td><td>13-676-10K</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile Disposable pipettes, all-plastic wrap, 5 ml</td><td>Fisherbrand</td><td>13-676-10H</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterilization Pouches, 19 x 33 cm</td><td>Crosstex</td><td>SCL</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Strainer, 40 &amp;micro;m</td><td>BD Falcon</td><td>352340</td><td></td></tr><tr><td>Tissue Culture Dishes, 60 mm</td><td>BD Falcon</td><td>351007</td><td></td></tr><tr><td>Tube Racks, Interlocking Four-Way</td><td>Fisherbrand</td><td>03-448-17</td><td></td></tr><tr><td>Water Bath</td><td>Fisherbrand</td><td>S52602Q</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Neural Progenitor Cell-Specific Processing Reagents</td><td></td><td></td><td></td></tr><tr><td>Neural Stem Cell Expansion Medium (Stemline)</td><td>Sigma-Aldrich</td><td>S3194-500ML</td><td>Important to use the Stemline brand</td></tr><tr><td>Recombinant Human Epidermal Growth Factor (EGF)</td><td>Millipore</td><td>GF316</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Recombinant Human Leukemia Inhibitory Factor (LIF)</td><td>Millipore</td><td>LIF1010</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Trypan Blue (0.4%)</td><td>Sigma-Aldrich</td><td>T8154-100ML</td><td>multiple manufacturers/suppliers</td></tr><tr><td>TrypLE Select (1x)</td><td>Life Technologies</td><td>12563-011</td><td></td></tr></tbody></table>

a cgmp-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue

Araştırma deneyleri ve klinik denemeler için tek bir örnekten hücrelerinin çok sayıda toplamak için bir hücre genişleme tekniği büyük ölçüde kök hücre topluluğuna yarar sağlayacak. Birçok mevcut genişleme yöntemler zahmetli ve masraflı ve tam ayrılma içeren bu birkaç kök ve progenitör hücre tipleri farklılaşmasını ya da erken yaşlanmayı geçmesi neden olabilir. Bu sorunların üstesinden gelmek için, basit ve ucuz olan &quot;kesme&quot; olarak adlandırılan bir otomatik mekanik pasajı bir yöntem geliştirdik. Bu teknik, tek bir hücre içine, kimyasal ya da enzimatik ayrışma önler ve bunun yerine sabit bir hücre / hücre teması muhafaza süspansiyon, küremsi kültürlerin büyük ölçekli bir genişleme sağlar. Doğrama yöntem öncelikle fetal beyin kaynaklı nöral progenitör hücreleri veya neurospheres süredir kullanılan ve son zamanlarda embriyonik ve uyarılmış pluripotent kök hücrelerinden elde edilen nöral kök hücreler ile kullanım için yayımlanmıştır. Prosedür involves bir doku kültürü Petri kabı üzerine neurospheres tohumlama ve sonradan etkin elle mekanik olarak her küre Ayrıştırıcı sıkıcı sürecini otomatize hücreleri aracılığıyla keskin, steril bir bıçak geçirerek. Kültür içinde hücreleri süspansiyon uygun bir yüzey alanı-hacim oranı sağlar; 500.000 &#39;den fazla hücre çapı en az 0.5 mm, tek neurosphere içinde yetiştirilebilir gibi. Bir T175 bir şişe içinde, 50000000 fazla hücreleri sadece 15000000 yapışık kültürlerinde göre süspansiyon kültürlerinde büyüyebilir. Önemlisi, kıyma prosedür klinik dereceli hücre ürünlerin kütle miktarı üretimine izin veren, güncel iyi üretim uygulamaları (cGMP) altında kullanılmıştır.

<img alt="Şekil 5," fo:content-width="5in" src="/files/ftp_upload/51219/51219fig5highres.jpg" width="500" /> P19&#39;un de donduruldu hNPCs Şekil 5. Temsilcisi veri. A) Öngörülen hücre sayıları, sonra çözülmüş ve kesme yöntemi ile pasajlanır neurospheres karşılaştırıldığında enzimatik ayrılma kullanarak yapışık tek tabaka olarak genişletti. Hücreler p20 eritildi edildiğinde Gün 0 gösterir. B) küreler Temsilcisi ...

Watch this Scientific Journal Video about A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue at JoVE.

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

Bu protokol, tek bir hücre süspansiyonu için ayrışma olmadan küresel nöral kök ve projenitör hücre agrega genişlemesini sağlayan bir yeni mekanik bir kesme yöntemi tarif etmektedir. Hücre / hücre teması sürdürmek üzerinde 40 geçişleri için hızlı ve istikrarlı bir büyüme sağlar.

İnsan Nöral Agregalarının bir cGMP uygulanabilir Açılım Yöntemi kök hücrelerin pluripotent kök hücreleri veya Fetal Beyin dokusundan türetilen

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

a-cgmp-applicable-expansion-method-for-aggregates-human-neural-stem

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

13.4K Görüntüleme.  Cedars-Sinai Medical Center.  Bu protokol, tek bir hücre süspansiyonu için ayrışma olmadan küresel nöral kök ve projenitör hücre agrega genişlemesini sağlayan bir yeni mekanik bir kesme yöntemi tarif etmektedir. Hücre / hücre teması sürdürmek üzerinde 40 geçişleri için hızlı ve istikrarlı bir büyüme sağlar. 

Video: A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Periventriküler Yetişkin Rat Bölgesinde ve İnsan Omurilik gelen Nöral Kök / Progenitör Hücreler izolasyonu

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Gelişim Biyolojisi

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

İnsan uyarılmış pluripotent kök hücrelerin türetilmiş Piramidal Nöronlar gelen Dendritik Spine Üç boyutlu Niceleme

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

İnsan Nöral Kök Hücreler Pasajlanması

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Biyoloji

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.

Atılan İnsan Fetal Kortikal Doku Sinir Kök Hücre Üretimi

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

Nörobilim

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Nöronal atalarıdır ve Nöronlar Verimli türetilmesi

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

iPSC türetilmiş İnsan Beyin Organoids oluşturulması Erken Nörogelişimsel Bozuklukları Model

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Nörotoksisite Testi İçin İnsan Kaynaklı Pluripotent Kök Hücrelerin Nöronlar ve Glia Karışık Kültürlerine Ayrıştırılması Protokolü

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Yetişkin İnsan Fibroblastları türetilmesi ve Genişletilebilir Sinir Progenitör Hücreler içine Doğrudan Dönüşüm

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Kan Türevi Nöronal Hücrelerin GM'den Arındırılmış Üretimi

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor.
In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

This article describes a protocol for the generation of human induced pluripotent stem cell-derived neurons in a benchtop 3D suspension bioreactor.

Üç Boyutlu Süspansiyon Biyoreaktöründe İnsan Nörojenin 2-İndüklenebilir Nöronların Üretimi

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first ...