Wir danken Dr. Soshana Svendsen für kritische Überprüfung und Bearbeitung des vorliegenden Berichts. Diese Arbeit wurde durch das NIH / NINDS 1U24NS078370-01 und CIRM DR2A-05320 zu beigetragen.

Die Autoren haben nichts zu offenbaren.

<img alt="Fig. 6" fo:content-width="5in" src="/files/ftp_upload/51219/51219fig6highres.jpg" width="500" /> Abbildung 6. Chopping Schema. Erweiterung Sphäroid Stammzellen / Vorläuferzellen in Kultur mit Hilfe der mechanischen Hackmethode. Kritische Schritte Eine Übersicht des Hackexpansions Paradigma ist in Fig. 6 gezeigt. HNPC Kugelgröße ist einer der wichtigen Kriterien, die bei Pa...

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

Es gibt eine lange Geschichte der Erweiterung Nagetier neuralen Stammzellen in Kultur entweder als Monoschicht oder allgemeiner Neurosphären 1-3 4-7. Zusätzlich wurden menschliche neurale Vorläuferzellen (hNPCs) aus verschiedenen Regionen des zentralen Nervensystems zu entwickeln 8-17 isoliert in vitro expandiert wurden. Diese Zellen sind bi-potent, und zur Differenzierung zu beiden Astrozyten und Neuronen und haben ein sehr nützliches Werkzeug bei der Untersuchung der neuralen Entwicklung 18,19 und 20,21 Krankheitsmechanismus. hNPCs sind auch in vielen verschiedenen Tiermodellen der Krankheit des zentralen Nervensystems mit unterschiedlichen Stufen der Integration, das Überleben und die funktionalen Wirkungen 22-24 verpflanzt worden. Oft epidermalen Wachstumsfaktor (EGF) und / oder Fibroblasten-Wachstumsfaktor-2 (FGF-2) 25-28 - - und beide haft 29 und drei Traditionell Nagetier oder menschliche fötale NSC an Wachstumsfaktoren ausgesetztSphäroid-dimensionalen Systemen werden typischerweise unter Verwendung der enzymatischen Spaltung in eine Einzelzellsuspension 30-34 agiert. Die Standard-Methode, um Zellen für die Forschung oder die klinische Anwendung zu erweitern ist als Anhänger Monoschicht durch einfache Manipulation. Wir haben jedoch gezeigt, dass die Passage Mono und Neurosphäre hNPCs mit enzymatische oder chemische Lösungen, führte im frühen Seneszenz 35. Darüber hinaus kann die enzymatische Spaltung bei erhöhten Differenzierung und karyotypischen Anomalien anhand von Daten mit embryonalen Stammzellen 36-38 gezeigt, führen. Obwohl die Standardmethode der Passage hat hNPCs aktuellen guten Herstellungspraxis (cGMP) hochwertige Produkte, die in Phase 1 der klinischen Studien (Stem Cells Inc., Neuralstem Inc.) gegangen produziert, erlaubt die Methode nur ein paar Runden Zellamplifikation, die Begrenzung der Bank Potenzial. Natürlich könnte große Forschungsexperimente und zukünftigen klinischen Studien von der Möglichkeit profitierenpropagieren Zellen in der Masse und mit verzögerter Seneszenz zu großen Wachstums-und Zellbanken zu ermöglichen. Um diesem Bedarf zu begegnen, haben wir ein neues und automatisierten Weise mechanisch Passage intakter Neurosphären durch &quot;Zerhacken&quot; sie in kleinen Clustern auf Zell-Zell-Kontakt. Dieses Verfahren stark erhöht deren Lebensdauer 39 und Suspensionskultur ermöglicht eine effizientere Nutzung der Inkubator Platz im Vergleich zu Monolayer-Kulturen, wie bei einem alternativen 3D-Bioreaktor-Kultur-Methode 40 gesehen. Die zur Verfügung gestellten Hack-Protokoll ermöglicht die Produktion von großen Banken von einem fetalen Probe größer als Durchgang 10, eine unwahrscheinliche Leistung mit Standard-Methoden Passagieren. Während diese Methode für das Passagieren hNPCs ist unkonventionell, wird er immer beliebter, und wurde vor kurzem mit anderen Zelltypen wie neuronale Stammzellen aus menschlichen embryonalen und induzierten pluripotenten Stammzellen, so dass groß angelegte Erweiterung für verschiedene Anwendungen, einschließlich in v veröffentlichtitro Krankheit Modellierung 41-46. Wichtig ist, dass ein cGMP-grade hNPC Zellbank bereits mit dem Schneideverfahren hergestellt wurde, zeigt, dass die Technik zur zukünftigen klinischen Anwendungen verwendet werden.

<table border="0" cellpadding="0" cellspacing="0" width="1230">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Beaker, 50 mL</td>
			<td style="width:159px;">Fisherbrand</td>
			<td>FB-100-50</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Bio-Safety Cabinet, class II</td>
			<td style="width:159px;">Baker</td>
			<td style="width:128px;">SG-603A</td>
			<td style="width:464px;">4 ft. or 6 ft. model.&#160; 6 ft. model recommended; multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Blades, Double-edge Prep</td>
			<td style="width:159px;">Personna</td>
			<td style="width:128px;">74-0002</td>
			<td style="width:464px;">multiple manufacturers/suppliers.&#160; CAUTION: Sharp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Cell Freezing Media</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:128px;">C6295-50ML</td>
			<td style="width:464px;">DMSO, serum-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Centrifuge, swing-bucket with 15 mL inserts</td>
			<td style="width:159px;">Eppendorf</td>
			<td style="width:128px;">5810 R</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Conical Tubes, 15 mL</td>
			<td style="width:159px;">Fisherbrand</td>
			<td style="width:128px;">S50712</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Conical Tubes, 50 mL</td>
			<td style="width:159px;">BD Falcon</td>
			<td style="width:128px;">352074</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Controlled Rate Freezer</td>
			<td style="width:159px;">Planer&#160;</td>
			<td style="width:128px;">Kryo 750</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Cryovials, 2 mL</td>
			<td style="width:159px;">Corning</td>
			<td style="width:128px;">430488</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Culture Flask, Vented, T12.5</td>
			<td style="width:159px;">BD Falcon</td>
			<td style="width:128px;">353107</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Culture Flask, Vented, T25</td>
			<td style="width:159px;">BD Falcon</td>
			<td style="width:128px;">353081</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Culture Flask, Vented, T175</td>
			<td style="width:159px;">BD Falcon</td>
			<td style="width:128px;">353045</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Culture Flask, Vented, T75&#160;</td>
			<td style="width:159px;">BD Falcon</td>
			<td style="width:128px;">353110</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Filter, 0.22 &#181;m, attached cup, 1 L</td>
			<td style="width:159px;">Millipore</td>
			<td style="width:128px;">SCGPU11RE</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Filter, 0.22 &#181;m, attached cup, 150 mL</td>
			<td style="width:159px;">Millipore</td>
			<td style="width:128px;">SCGVU01RE</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Filter, 0.22 &#181;m, attached cup, 500 mL</td>
			<td style="width:159px;">Millipore</td>
			<td style="width:128px;">SCGPU05RE</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Filter, 0.22 &#181;m, attached cup, 50 mL</td>
			<td style="width:159px;">Millipore</td>
			<td style="width:128px;">SCGP00525</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Filter Paper, 8.5 cm circles</td>
			<td style="width:159px;">Whatman/GE</td>
			<td style="width:128px;">1001-085</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Forceps, Standard Pattern - Serrated/Curved/18 cm</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:128px;">11001-18</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Freezing Chamber, Isopropyl Alcohol</td>
			<td style="width:159px;">Nalgene</td>
			<td style="width:128px;">5100-0001</td>
			<td style="width:464px;">&#34;Mr. Frosty&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Incubator, 37&#176;C/5% CO2</td>
			<td style="width:159px;">Forma</td>
			<td style="width:128px;">370 series</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Hemacytometer, Phase</td>
			<td style="width:159px;">Hausser Scientific</td>
			<td style="width:128px;">1475</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">McIlwain Tissue Chopper</td>
			<td style="width:159px;">Lafayette Instruments</td>
			<td style="width:128px;">TC752-PD</td>
			<td style="width:464px;">&#160;Petri dish modification required.&#160;&#160; CAUTION:&#160; Moving, sharp blade.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Micropipettor, 1 - 10 &#956;L</td>
			<td style="width:159px;">Gilson</td>
			<td style="width:128px;">F144562</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Micropipettor, 100 - 1000 &#956;L (starter kit)</td>
			<td style="width:159px;">Gilson</td>
			<td style="width:128px;">F167700</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Micropipettor, 2 - 20 &#956;L (starter kit)</td>
			<td style="width:159px;">Gilson</td>
			<td style="width:128px;">F167700</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Micropipettor, 20 - 200 &#956;L (starter kit)</td>
			<td style="width:159px;">Gilson</td>
			<td style="width:128px;">F167700</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nutdriver, Autoclavable, 5/16&#34;</td>
			<td style="width:159px;">Steritool</td>
			<td style="width:128px;">10302</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur Pipets, cotton-plugged</td>
			<td style="width:159px;">Fisherbrand</td>
			<td style="width:128px;">13-678-8B</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Petri Dish, Glass, Autoclavable</td>
			<td style="width:159px;">Corning</td>
			<td style="width:128px;">3160-100</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Pipet Aid</td>
			<td style="width:159px;">Drummond</td>
			<td style="width:128px;">4-000-101</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Shim disc</td>
			<td style="width:159px;">McMaster-Carr</td>
			<td style="width:128px;">VARIABLE</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Sterile barrier pipet tips, 10 &#956;L</td>
			<td style="width:159px;">AvantGuard</td>
			<td style="width:128px;">AV10R-H</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Sterile barrier pipet tips, 1000 &#956;L</td>
			<td style="width:159px;">AvantGuard</td>
			<td style="width:128px;">AV1000</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Sterile barrier pipet tips, 20 &#956;L</td>
			<td style="width:159px;">AvantGuard</td>
			<td style="width:128px;">AV20-H</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Sterile barrier pipet tips, 200 &#956;L</td>
			<td style="width:159px;">AvantGuard</td>
			<td style="width:128px;">AV200-H</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Sterile Disposable pipettes, all-plastic wrap, 10 mL</td>
			<td style="width:159px;">Fisherbrand</td>
			<td style="width:128px;">13-676-10J</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Sterile Disposable pipettes, all-plastic wrap, 2 mL</td>
			<td style="width:159px;">Fisherbrand</td>
			<td style="width:128px;">13-675-3C</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Sterile Disposable pipettes, all-plastic wrap, 25 mL</td>
			<td style="width:159px;">Fisherbrand</td>
			<td style="width:128px;">13-676-10K</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Sterile Disposable pipettes, all-plastic wrap, 5 mL</td>
			<td style="width:159px;">Fisherbrand</td>
			<td style="width:128px;">13-676-10H</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Sterilization Pouches, 19 x 33 cm</td>
			<td style="width:159px;">Crosstex</td>
			<td style="width:128px;">SCL</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:479px;">Strainer, 40 &#181;m</td>
			<td style="width:159px;">BD Falcon</td>
			<td style="width:128px;">352340</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Tissue Culture Dishes, 60 mm</td>
			<td style="width:159px;">BD Falcon</td>
			<td style="width:128px;">351007</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Tube Racks, Interlocking Four-Way</td>
			<td style="width:159px;">Fisherbrand</td>
			<td style="width:128px;">03-448-17</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Water Bath</td>
			<td style="width:159px;">Fisherbrand</td>
			<td>S52602Q</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;"></td>
			<td style="width:159px;"></td>
			<td></td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Neural Progenitor Cell-Specific Processing Reagents</td>
			<td style="width:159px;"></td>
			<td style="width:128px;"></td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Neural Stem Cell Expansion Medium (Stemline)</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:128px;">S3194-500ML</td>
			<td style="width:464px;">Important to use the Stemline brand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Recombinant Human Epidermal Growth Factor (EGF)</td>
			<td style="width:159px;">Millipore</td>
			<td style="width:128px;">GF316</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Recombinant Human Leukemia Inhibitory Factor (LIF)</td>
			<td style="width:159px;">Millipore</td>
			<td style="width:128px;">LIF1010</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Trypan Blue (0.4%)</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:128px;">T8154-100ML</td>
			<td style="width:464px;">multiple manufacturers/suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">TrypLE Select (1X)</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:128px;">12563-011</td>
			<td style="width:464px;"></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

Ein Zellexpansion Technik, um eine große Zahl von Zellen, die aus einer einzelnen Probe für Forschungsexperimente und klinische Studien sammeln würde die Stammzell Gemeinschaft profitieren. Viele aktuelle Erweiterung Methoden sind aufwendig und teuer, und solche, die vollständige Dissoziation kann dazu führen, mehrere Stamm-und Vorläuferzelltypen, die Differenzierung oder frühen Seneszenz. Um diese Probleme zu überwinden, haben wir einen automatisierten mechanischen Passagieren Verfahren als &quot;Hacken&quot;, das einfach und kostengünstig ist bezeichnet entwickelt. Diese Technik vermeidet chemische oder enzymatische Dissoziation in Einzelzellen und ermöglicht die großflächige Erweiterung des suspendiert, Sphäroid-Kulturen, die konstant Zell / Zell-Kontakt zu halten, statt. Die Schneideverfahren hat sich vor allem für fötale Gehirn-abgeleiteten neuralen Vorläuferzellen oder Neurosphären verwendet wurden, und wurde kürzlich für die Verwendung mit neuronalen Stammzellen aus embryonalen und induzierten pluripotenten Stammzellen veröffentlicht. Das Verfahren involves Aussaat Neurosphären auf einer Gewebekultur Petrischale und anschließend vorbei einen scharfen, sterilen Messer durch die Zellen effektiv die Automatisierung der mühsame Prozess der manuell mechanisch distanziert jede Kugel. Suspendieren der Zellen in der Kultur ein günstiges Oberflächen-zu-Volumen-Verhältnis; wie mehr als 500.000 Zellen können in einer einzelnen Neuro von weniger als 0,5 mm im Durchmesser wachsen. In einem Kolben T175 kann über 50 Millionen Zellen in Suspensionskulturen wachsen im Vergleich zu nur 15 Millionen in adhärenten Kulturen. Wichtig ist, dass das Hackverfahren gemäß der aktuellen guten Herstellungspraxis (cGMP) eingesetzt, wodurch Massenmenge Produktion von klinischem Zellprodukte.

<img alt="Figur 5" fo:content-width="5in" src="/files/ftp_upload/51219/51219fig5highres.jpg" width="500" /> Abbildung 5. Repräsentative Daten. A) Projizierte Zellzahlen von hNPCs an p19 eingefroren, aufgetaut und dann als Anhänger Monolayer mit enzymatischen Dissoziation im Vergleich zu Neurosphären über den Hack Verfahren agiert erweitert. Tag 0 stellt, wenn die Zellen aufgetaut p20 bei B.) Repräsentative Bilder von Kugeln vor, hacken, 10...

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

Dieses Protokoll beschreibt einen neuartigen mechanischen Schneideverfahren, das die Erweiterung der sphärischen neuralen Stamm-und Vorläuferzellaggregate ohne Dissoziation zu einer Einzelzellsuspension ermöglicht. Die Aufrechterhaltung Zell / Zell-Kontakt ermöglicht eine schnelle und stabile Wachstum seit über 40 Passagen.

Ein cGMP-Verfahren zur Erweiterung anwendbar Aggregate von humanen neuralen Stamm-und Vorläuferzellen, abgeleitet von pluripotenten Stammzellen oder fetalen Hirngewebe

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

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Research

JoVE Journal

Neuroscience

13.4K Aufrufe.  Cedars-Sinai Medical Center.  Dieses Protokoll beschreibt einen neuartigen mechanischen Schneideverfahren, das die Erweiterung der sphärischen neuralen Stamm-und Vorläuferzellaggregate ohne Dissoziation zu einer Einzelzellsuspension ermöglicht. Die Aufrechterhaltung Zell / Zell-Kontakt ermöglicht eine schnelle und stabile Wachstum seit über 40 Passagen. 

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

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.

Neural-Colony Forming Zell-Assay: Ein Test zur Bona Fide neuralen Stammzellen aus neuralen Vorläuferzellen Discriminate

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). ...

Forschung

Neurowissenschaften

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.

Generierung von neuralen Stammzellen aus menschlichen Föten Ausrangierte Rindengewebe

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

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-Cell-Imaging von Sinnesorgan Vorläuferzellen im Intact Drosophila Puppen

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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.

Isolierung und Kultivierung von Zellen der Neuralleiste aus embryonalen murinen Neuralrohr

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

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Progenitor-abgeleiteten Oligodendrozyten Culture System von Human Fetal Gehirn

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

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.

Feeder freie Ableitung von Neural Crest Vorläuferzellen aus menschlichen pluripotenten Stammzellen

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

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Regie dopaminergen Neuron Differenzierung von humanen pluripotenten Stammzellen

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

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

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identifizieren von Zelloberflächenmarkern von primären neuralen Stamm- und Vorläuferzellen durch metabolische Kennzeichnung von Sialoglycan

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

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Ableitung, Expansion, Kryokonservierung und Charakterisierung von mikrovaskulären Endothelzellen des Gehirns aus humaninduzierten Pluripotenten Stammzellen

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Durchflusszytometrische Analyse multipler mitochondrialer Parameter in humaninduzierten pluripotenten Stammzellen und deren neuronalen und glialen Derivaten

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...