Name: Een op live-cell beelden gebaseerde machine learning-strategie om pluripotente stamceldifferentiatie te monitoren (Video) | JoVE Journal
Uploaded: 2024-10-04T13:30:00.000+00:00
Duration: 11 min 38 s
Description: Leer een machine learning-strategie voor het monitoren van pluripotente stamceldifferentiatie met live-cell beelden. Deze niet-invasieve methode optimaliseert differentiatieprotocollen en verbetert de reproduceerbaarheid voor cardiale weefselengineering.

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<meta charset="utf8"></head><body>Monitoring van pluripotente stamceldifferentiatie door middel van op afbeeldingen gebaseerde ML-strategie

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<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>25200056</td><td>Verdunde digests werden gebruikt voor CPC en CM-digestie</td></tr><tr><td>4% Paraformaldehyde in PBS</td><td>KeyGEN BioTECH</td><td>KGIHC016</td><td></td></tr><tr><td>6-well Cell Culture Plate</td><td>NEST</td><td>703001</td><td></td></tr><tr><td>96-well Cell Culture Plate</td><td>NEST</td><td>701001</td><td></td></tr><tr><td>B27 Supplement</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>B27 Supplement Minus Insulin</td><td>Gibco</td><td>A1895601</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>GPC BIOTECH</td><td>AA904-100G</td><td></td></tr><tr><td>Celldiscoverer 7</td><td>Zeiss</td><td></td><td>Instrumenten die worden gebruikt om heldere-veldbeelden te maken tijdens differentiatie en uiteindelijke cTnT-beelden</td></tr><tr><td>CHIR99021</td><td>Selleck</td><td>S1263</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>12634010</td><td></td></tr><tr><td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo</td><td>A-21202</td><td>Secundaire antilichaam</td></tr><tr><td>FACSAria III</td><td>BD Biosciences</td><td></td><td>Flow cytometry sorter</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>VISTECH</td><td>SE100-B</td><td></td></tr><tr><td>Hoechst 33342</td><td>YEASEN</td><td>40732ES03</td><td></td></tr><tr><td>Human Pluripotent Stem Cell Chemical-defined Medium</td><td>Cauliscell Inc</td><td>400105</td><td>Basismedium van PSC-voorbereidingsmedium</td></tr><tr><td>iPS-18</td><td>TaKaRa</td><td>Y00300</td><td></td></tr><tr><td>iPS-B1</td><td>Cellapy</td><td>CA4025106</td><td></td></tr><tr><td>iPS-F</td><td>Nuwacell</td><td>RC01001-B</td><td></td></tr><tr><td>iPS-M</td><td>Nuwacell</td><td>RC01001-A</td><td></td></tr><tr><td>IWR1-1-endo</td><td>Selleck</td><td>S7086</td><td>IWR1</td></tr><tr><td>Jupyter Notebook</td><td>N/A</td><td>Versie 6.4.0</td><td><a href="https://jupyter.org/">https://jupyter.org/</a></td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>Versie R2020a</td><td>Software voor wetenschappelijke berekening en afbeeldingsannotatie</td></tr><tr><td>Matrigel Matrix</td><td>Corning</td><td>354230</td><td>Matrigel</td></tr><tr><td>Mouse monoclonal IgG1 anti-cTnT</td><td>Thermo</td><td>MA5-12960</td><td>cTnT primaire antilichaam</td></tr><tr><td>Normal Donkey Serum</td><td>Jackson</td><td>017-000-121</td><td></td></tr><tr><td>ORCA-Flash 4.0 V3 digital CMOS camera</td><td>Hamamatsu</td><td>C13440-20CU</td><td>De digitale camera geassembleerd op Celldiscoverer7</td></tr><tr><td>PBS</td><td>NEB</td><td>21-040-CVR</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Pluripotency Growth Mater 1 basal medium</td><td>Cellapy</td><td>CA1007500-1</td><td>Basismedium van PSC-kweekmedium</td></tr><tr><td>Pluripotency Growth Mater 1 supplement</td><td>Cellapy</td><td>CA1007500-2</td><td>Supplement van PSC-kweekmedium</td></tr><tr><td>Prism</td><td>Graphpad</td><td>Versie 8/9</td><td>Statistisch software voor statistische analyse en plotten</td></tr><tr><td>Python</td><td>N/A</td><td>versie 3.6</td><td>Python 3-omgeving voor wetenschappelijke berekening, met packages pytorch (1.9.0), numpy, scipy, pandas, visdom, scikit-learn, scikit-image, opencv-python en matplotlib software voor wetenschappelijke berekening en afbeeldingsannotatie.</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875176</td><td></td></tr><tr><td>Supplement hPSC-CDM (500x)</td><td>Cauliscell Inc</td><td>00015</td><td>Supplement van PSC-voorbereidingsmedium</td></tr><tr><td>TiE</td><td>Nikon</td><td></td><td>Een omgekeerde fluorescentiemicroscoop (met wijzigingen) voor region-selevtive zuivering</td></tr><tr><td>Triton&nbsp;X-100</td><td>Amresco</td><td>9002-93-1</td><td></td></tr><tr><td>Versene Solution</td><td>Thermo</td><td>15040066</td><td>EDTA-oplossing voor PSC-digestie</td></tr><tr><td>Y27632</td><td>Selleck</td><td>S6390</td><td></td></tr><tr><td>Zen</td><td>Zeiss</td><td>Versie 3.1</td><td>Een ondersteunende software van Celldiscoverer7 voor&nbsp; beeldverwerving, verwerking en analyse</td></tr></tbody></table>

een op live-cell beelden gebaseerde machine learning-strategie om pluripotente stamceldifferentiatie te monitoren

A Live-cell Image-Based Machine Learning Strategy to Monitor Pluripotent Stem Cell Differentiation

Een op live-cell beelden gebaseerde machine learning-strategie om pluripotente stamceldifferentiatie te monitoren

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available ...

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Source: Gómez-Villafuertes, R., et. al., Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations. J. Vis. Exp. (2017) The video demonstrates the process of preparing, imaging, and tracking adult neural stem cells in real time to study cellular behaviors, including division and differentiation.

 Live Imaging and Single-Cell Tracking to Monitor Neural Lineage in Adult Neural Stem Cells

Het protocol in dit onderzoek beschrijft werkwijzen voor de real-time monitoring herprogrammering progressie via de kinetische meting van positieve en negatieve pluripotente stamcel markers gebruikt flowcytometrie analyse. Het protocol bevat ook de imaging-gebaseerde beoordeling van de morfologie, en merker of reporter expressie tijdens iPSC generatie.

Kinetic Meet- en Real Time Visualisatie van Somatic herprogrammeren

Een robuust protocol controleren neurale populaties time-lapse video-microscopie gevolgd door softwarematige nabewerking is beschreven. Deze methode geeft een krachtig hulpmiddel voor het identificeren van biologische gebeurtenissen in een geselecteerde populatie tijdens live imaging experimenten.

Live Imaging gevolgd door eencellige bijhouden van de biologie van de cel van de Monitor en de progressie van de bloedlijn van meerdere neurale populaties

We tonen de automatisering van door de mens geïnduceerde pluripotente stamcelculturen (hiPSC) culturen en neuronale differentiaties die compatibel zijn met geautomatiseerde beeldvorming en analyse.

Geautomatiseerde productie van menselijke geïnduceerde pluripotente stamcel-afgeleide corticale en dopaminerge neuronen met geïntegreerde live-cel monitoring

Beschrijven we een klein molecuul-gebaseerd protocol voor de differentiatie van muis embryonale stamcellen in de oligodendrocyt voorloper cellen (OPC's). Dit protocol genereert Olig2 + NG2 + OPC's met een hoge efficiëntie met 30 dagen van differentiatie. We beschrijven ook een methode om "spike" OPC's die kunnen vuren actiepotentialen genereren.

Differentiatie van embryonale stamcellen in de oligodendrocyt Voorlopers

Dit document heeft tot doel de lezer in de exploitatie van een geïntegreerde atomic force-microscoop optische beeldvorming instrueren voor de mechanische stimulatie van levende cellen in cultuur. Een stap-voor-stap protocol wordt gepresenteerd. Een representatieve dataset die levende cellen als reactie op mechanische stimulatie shows gepresenteerd.

Live-cel respons op mechanische stimulatie onderzocht door Integrated Optical en Atomic Force Microscopy

Ondanks de recente ontwikkelingen in genetische modificatie, transfectie van humane embryonale stamcellen (hESC 's) blijft een grillig proces. Voor zover wij weten, zijn systematische en efficiënte methoden om menselijke geïnduceerde pluripotente stamcellen (iPSCs) transfecteren niet gemeld. Hier beschrijven we robuuste protocollen om efficiënt te transfecteren en nucleofect menselijke iPSCs.

Transfecteren en Nucleofecting Human geïnduceerde pluripotente stamcellen

Een methode om de mens geïnduceerde pluripotente stamcellen (iPSCs) via retrovirus-gemedieerde ectopische expressie van OCT4 genereren, wordt SOX2, KLF4 en MYC beschreven. Een praktische manier om de menselijke IPSC kolonies op basis van GFP expressie te identificeren wordt ook besproken.

Herprogrammeren menselijke somatische cellen in geïnduceerde pluripotente stamcellen (iPSCs) Met behulp van retrovirale vector met GFP

Pluripotente stamcellen, hetzij embryonale of geïnduceerde pluripotente stamcellen (iPS) cellen vormen een waardevolle bron van menselijke gedifferentieerde cellen, zoals cardiomyocyten. Hier zullen we ons richten op de cardiale inductie van iPS cellen, laten zien hoe ze te gebruiken om functionele menselijke hartspiercellen te verkrijgen via een embryoid lichamen-gebaseerd protocol.

Generatie van Human Cardiomyocytes: Een Differentiatie Protocol van Feeder-vrije Human geïnduceerde pluripotente stamcellen

Time-lapse microscopie van fluorescent gelabelde autofagie markers zorgt voor de opvolging van de dynamische autofagie respons met een hoge temporele resolutie. Met specifieke autofagie en organel markers in een combinatie van 3 verschillende kleuren, kunnen we de bijdrage van een eiwit autophagosome vorming in een robuuste ruimtelijke en temporele context volgen.

Live cell imaging van Vroege Autophagy Evenementen: Omegasomes en Beyond

Leer een machine learning-strategie voor het monitoren van pluripotente stamceldifferentiatie met live-cell beelden. Deze niet-invasieve methode optimaliseert differentiatieprotocollen en verbetert de reproduceerbaarheid voor cardiale weefselengineering.

Een op live-cell beelden gebaseerde machine learning-strategie om pluripotente stamceldifferentiatie te monitoren

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