Name: in vitro differentiation of human mesenchymal stem cells into functional cardiomyocyte-like cells
Uploaded: 2017-08-09T12:30:00
Description: Watch this Scientific Journal Video about In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells at JoVE.com

Forfatterne takke de følgende medarbejdere og forskning personale for deres bidrag: Matthew Librach, Leila Maghen, Tanya A. Baretto, Shlomit Kenigsberg og Andrée Gauthier-Fisher. Dette arbejde blev støttet af den The Ontario forskningsfond - Research Excellence (ORF-RE, runde #7) og oprette programmet Inc.

All studies involving animals were conducted and reported according to ARRIVE guidelines48. All studies were performed with institutional research ethics board approval (REB number 454-2011, Sunnybrook Research Institute; REB 29889, University of Toronto, Toronto, Canada). All animal procedures were approved by the Animal Care Committee of the University Health Network (Toronto, Canada), and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, 8...

Hjerte differentieringen af stamceller har været under udvikling i 2 årtier, med flere forskellige strategier, der anvendes til at generere cardiomyocyte-lignende celler fra MSC kilder. Mange af disse strategier, men er ineffektiv, og de betingelser der ofte ikke er repræsentative miljø transplanterede celler møde i vivo.
I modsætning til eksisterende metoder benytter protokollen præsenteres her en kombination af primære hjerte feeder lag og MSC samlede dannelse. De primære h...

Congestive hjerteinsufficiens (CHF) fortsætter som en førende årsag til sygelighed og dødelighed på verdensplan. CHF opstår ofte efter det massive tab af cardiomyocytes og udviklingen af celle-fri arvæv som patologiske resultatet af et myokardieinfarkt (MI)1. Mens hjertet er en delvist selvstændig fornyelse orgel, mindsker bosiddende stilk og stamfader celle poolen ansvarlig for udførelsen vævsregeneration betydeligt i overflod og funktion hos ældre patienter, ofte bliver utilstrækkelig til optimal opsving efter skade. Der er således stor interesse i at udvikle eksperimentelle behandlinger, der involverer transplantation af sunde donor celler i den beskadigede myokardiet. Det er bydende nødvendigt, at donor celler ikke kun genoprette struktur af væv, men også opnå en funktionel genopretning af de berørte myokardiet.
De indfødte hjerte beskæftiger hjerte væv-beboer og endogene stammer fra knoglemarv stamceller til efter skade reparere2,3,4. Regenerativ celler-host - og donor-stammer både-skal have kapacitet til at indhente passende fænotype og funktion i mikromiljø i remodeling myokardiet, sammen med evnen til at effektivt og sikkert erstatte de tabte celler. In vitro differentiering metoder er blevet brugt flittigt til at opnå høj effektivitet, stamcelle-baseret cardiomyocyte produktion5,6. Profilen udtryk af cardiac lineage markører bruges til at definere processen med stamcelle differentiering mod hjerte lineage7. Tidlig differentiering markører, såsom NKX2.5, myocyte forstærker faktor 2 C (Mef2c), og GATA48,9, kan være en indikation af indledningen af cardiomyogenic processen. Modne cardiomyocyte markører almindeligt anvendt til at vurdere differentiering effekten er signal regulerende protein α (SIRPA)10, cardiac troponin T (cTnT)11, tunge kæde hjerte myosin (MYH6)8,12,13og connexin 43 (Cx43)14,15,16. De metoder, der bruger embryonale stamceller (råd) og pluripotente stamceller (PSC) er blevet grundigt optimeret og drøftet med hensyn til detaljerne i induktive faktorer, ilt og næringsstoffer forløb, og den nøjagtige timing af aktion5,6,7,17,18. Ikke desto mindre præsentere ESC - og PSC-baserede teknologier stadig flere etiske og sikkerhed bekymringer, sammen med suboptimal elektrofysiologiske og immunologiske funktioner19,20. Værter transplanteret med disse celler ofte opleve immunorejection og kræve permanent immunosuppression. Dette er hovedsagelig på grund af misforholdet store histocompatibility complex (MHC) molekyler i host og donoren og til den resulterende T-celle respons21. Mens enkelte MHC klasse I matchende er en mulig løsning, en mere tilgængelige kliniske praksis ville kræve en celle kilde, der er universelt immunoprivileged at overvinde bekymring for afvisning.
Som en alternativ celle kilde til brug i kliniske applikationer, MSC'er, navnlig BMSCs, er blevet undersøgt til brug i væv revitalisering siden deres indledende beskrivelse i 199522. MSC'er menes at være bosat regenerativ celler, der kan findes i næsten enhver vaskulariserede væv23. Ved isoleret fra den ønskede kilde, MSCs kan nemt udvides i kultur, har omfattende paracrine kapacitet, og ofte har immunoprivileged eller immunmodulerende egenskaber24,25. Deres sikkerhed og effektivitet har allerede været vist i flere prækliniske undersøgelser, navnlig for kardiale regenerering3,26.
Mange MSC differentiering strategier udnytte farmakologiske stoffer, såsom 5-azacytidine22 og DMSO27, og vækst eller morphogenic faktorer, såsom BMP5,7,28,29 eller angiotensin-II30, med variabel effektivitet. Disse strategier, er imidlertid ikke baseret på de hindringer, som en naiv regenerativ celle er sandsynlig hen til opgør efter homing eller at blive leveret til stedet, skaden in vivo. Mere fysiologisk relevante strategier, mens vanskeligere at definere og manipulere, er baseret på den forudsætning, at MSC differentiering kan være fremkaldt via signaler fra væv mikromiljø, selv. Tidligere undersøgelser har vist, at eksponering for kardiale celle lysates31 eller ventrikulære myokardiet32,33, eller direkte kontakt med primære cardiomyocytes in vitro-15,34, kan øge udtryk for hjertets markører i MSCs. Andre har demonstreret spontane cardiomyogenesis efter behandling af kardiale skader med MSCs35,36,37,38, selv om delvis fusion af BMSCs og cardiomyocytes39,40 genereret den spirende myokardiet. Til vores viden, har funktionelle, spontant ordregivende cardiomyocytes fra menneskelige MSCs (hMSCs) af enhver væv kilde ikke endnu blevet rapporteret.
Den nuværende konsensus er, at alle MSCs opstår fra perivascular celler23. Unge MSCs med pericyte egenskaber kan isoleres fra regionen perivascular af menneskelige navlestreng væv41,42,43. I forhold til BMSCs besidder HUCPVCs øget differentiering potentielle og flere andre regenerativ fordele, både in vitro-41,44 og i vivo45,46,47. Især, har kilden er grænsefladen maternel-føtal, HUCPVCs betydeligt lavere immunogenicitet i forhold til voksne kilder til MSCs. Vores forskning fokuserer på karakterisering og præ-kliniske anvendelser af FTM HUCPVCs, den yngste kilde til MSCs undersøgt, som vi tidligere har vist sig at have øget proliferativ og højere multilineage differentiering kapacitet, herunder i cardiomyogenic slægt41.
Vi præsenterer her, en protokol, der kombinerer samlede dannelse og primære hjerte cellelag med feeder som induktive styrker at opnå komplet cardiomyogenic differentiering af MSC'er. aggregater giver en 3D-miljø, som bedre modeller betingelser i vivo i forhold til 2D vedhængende kulturer. Udnytte hjerte feeder lag skaber et miljø, der er repræsentativ for den ultimative transplantation site for at MSC'er. Vi demonstrere, at yngre kilder af MSC'er isoleret fra før eller efter fødslen umbilical snore har en højere kapacitet form aggregater og nå frem til hjerte fænotype sammenlignet med voksne BMSCs, mens den stadig vedholdt deres immun-privilegium. Udover den stejle højde af cardiac lineage markørgener og den inducerede udtryk af intracellulære (dvs. cTnT og MYH6) og celle-overflade proteiner (dvs., SIRPA og Cx43) specifikt for cardiomyocytes, vi viser, at differentiering af FTM HUCPVCs potentiale kan udnyttes med denne metode, og at de kan give anledning til spontant kontraherende cardiomyocyte-lignende celler.

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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">0.25% Trypsin/EDTA</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:119px;">25200056</td>
			<td style="width:299px;">For cell dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Alpha-MEM</td>
			<td style="width:203px;">Gibco</td>
			<td>12571071</td>
			<td style="width:299px;">For HUCPVC and BMSC culture media.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">PE-conjugated anti-human/mouse CD49f antibody</td>
			<td style="width:203px;">Biolegend</td>
			<td style="width:119px;">313612</td>
			<td style="width:299px;">Integrin marker for FC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">APC-conjugated human Cx43/GJA1 antibody</td>
			<td style="width:203px;">R&#38;D Systems</td>
			<td style="width:119px;">FAB7737A</td>
			<td style="width:299px;">Connexin 43 marker for FC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">FITC-conjugated HLA-A2 antibody</td>
			<td style="width:203px;">Genway Biotech Inc.</td>
			<td style="width:119px;">GWB-66FBD2</td>
			<td style="width:299px;">Immunogenicity marker for FC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">FITC-conjugated anti-HLA-G [MEM-G/9] antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:119px;">ab7904</td>
			<td style="width:299px;">Immunogenicity marker for FC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">FITC-conjugated mouse anti-human SIRPA/CD172a antibody</td>
			<td style="width:203px;">AbD Serotec/Bio-Rad</td>
			<td style="width:119px;">MCA2518F</td>
			<td style="width:299px;">Cardiac marker for FC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">APC-conjugated human TRA-1-85/CD147 antibody</td>
			<td style="width:203px;">R&#38;D Systems</td>
			<td style="width:119px;">FAB3195A</td>
			<td style="width:299px;">Human cell marker for FC and FACS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">FITC-conjugated human TRA-1-85/CD147 antibody</td>
			<td style="width:203px;">R&#38;D Systems</td>
			<td style="width:119px;">FAB3195F</td>
			<td style="width:299px;">Human cell marker for FC and FACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Anti-connexin 43/GJA1 antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:119px;">ab11370</td>
			<td style="width:299px;">Cx43. For ICC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Goat anti-rabbit IgG (H+L) cross-absorbed secondary antibody, Alexa Fluor 555</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:119px;">A-21428</td>
			<td style="width:299px;">For ICC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Anti-sarcomeric alpha actinin [EA-53] antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:119px;">ab9465</td>
			<td style="width:299px;">aSARC. For ICC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:297px;">Goat anti-mouse IgM heavy chain cross-absorbed secondary antibody, Alexa Fluor 555</td>
			<td style="width:203px;">Life Technologies</td>
			<td>A-21426</td>
			<td style="width:299px;">For ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Mef2C (D80C1) XP rabbit antibody</td>
			<td style="width:203px;">New England BioLabs Ltd.</td>
			<td style="width:119px;">5030S</td>
			<td style="width:299px;">For ICC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:119px;">A-21206</td>
			<td style="width:299px;">For ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Anti-nuclei (HuNu) (clone 235-1) antibody</td>
			<td style="width:203px;">EMD Millipore</td>
			<td style="width:119px;">MAB1281</td>
			<td style="width:299px;">For ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">MZ9.5 Stereomicroscope</td>
			<td style="width:203px;">Leica</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">For imaging aggregates.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">1.5 ml centrifuge microtubes</td>
			<td style="width:203px;">Axygen</td>
			<td style="width:119px;">MCT-150-C</td>
			<td style="width:299px;">For staining MSCs with fluorescent dye.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">ImageJ</td>
			<td style="width:203px;"></td>
			<td style="width:119px;"></td>
			<td style="width:299px;">Open source image processing software.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Aria II&#160;</td>
			<td style="width:203px;">BD</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">UHN SickKids FC Facility. For cell sorting.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Bone marrow mesechymal stromal cells</td>
			<td style="width:203px;">Lonza</td>
			<td style="width:119px;">PT-2501</td>
			<td style="width:299px;">BMSCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Bovine serum albumin</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7030-100G</td>
			<td style="width:299px;">BSA. To prepare solutions for ICC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:297px;">BrdU</td>
			<td style="width:203px;">EMD Millipore</td>
			<td style="width:119px;">MAB3424</td>
			<td style="width:299px;">Caution: BrdU is a strong teratogen and suspected mutagen. Please ensure proper training and refer to the SDS before use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Canto II</td>
			<td style="width:203px;">BD</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">UHN SickKids FC Facility. For flow cytometry.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">cDNA EcoDry Premix</td>
			<td style="width:203px;">Clontech/Takara</td>
			<td style="width:119px;">639570</td>
			<td style="width:299px;">For preparation of cDNA for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">CellTracker Green CMFDA Dye</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:119px;">C7025</td>
			<td style="width:299px;">Fluorescent imaging of cell cytoplasm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Countess automated cell counter</td>
			<td style="width:203px;">Invitrogen Inc.</td>
			<td style="width:119px;">C10227</td>
			<td style="width:299px;">For cell counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">DMEM-F12</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6421</td>
			<td style="width:299px;">For rat primary cardiomyocyte culture medium.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:297px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:203px;">Gibco</td>
			<td>10010023</td>
			<td style="width:299px;">D-PBS, without Ca2+, Mg2+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">EVOS</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">In-house fluorescent microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">FACSCalibur</td>
			<td style="width:203px;">BD</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">In-house. For flow cytometry.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Fetal bovine serum (Hyclone)</td>
			<td style="width:203px;">GE Healthcare</td>
			<td style="width:119px;">SH3039603</td>
			<td style="width:299px;">FBS. Component of cell culture medium.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:297px;">IDT Prime Time qPCR probes</td>
			<td style="width:203px;">Integrated Data Technologies</td>
			<td style="width:119px;">FAM fluorophore</td>
			<td style="width:299px;">http://www.idtdna.com/pages/products/gene-expression/primetime-qpcr-assays-and-primers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Lab Vision PermaFluor Aqueous Mounting Medium</td>
			<td style="width:203px;">ThermoScientific</td>
			<td style="width:119px;">TA-030-FM</td>
			<td style="width:299px;">For storage of cells to undergo ICC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">LSR II&#160;</td>
			<td style="width:203px;">BD</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">UHN SickKids FC Facility. For flow cytometry.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">MoFlo Astrios</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">UHN SickKids FC Facility. For cell sorting.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Normal goat serum</td>
			<td style="width:203px;">Cell Signaling Technology</td>
			<td style="width:119px;">5425S</td>
			<td style="width:299px;">NGS. Used in blocking solution for ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Nunc Lab-Tek II Chamber Coverglass, 8-wells</td>
			<td style="width:203px;">Thermo Scientific Nunc</td>
			<td style="width:119px;">155409</td>
			<td style="width:299px;">To prepare samples for ICC</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:297px;">OmniPur Triton X-100 Surfactant</td>
			<td style="width:203px;">EMD Millipore</td>
			<td style="width:119px;">9410-OP</td>
			<td style="width:299px;">As a component of permeabilizing solution when preparing cells for ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Paraformaldehyde, 16% Solution, EM Grade</td>
			<td style="width:203px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td style="width:299px;">For fixing cells for ICC.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Penicillin/streptomycin</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:119px;">15140122</td>
			<td style="width:299px;">Component of cell culture medium.</td>
		</tr>
		<tr height="110">
			<td height="110" style="height:110px;width:297px;">Primers</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:119px;">Custom Standard DNA Oligos, Desalted, 0.2 &#956;mol</td>
			<td style="width:299px;">CTnT_F: GGC AGC GGA AGA GGA TGC TGA A; CTnT_R: GAG GCA CCA AGT TGG GCA TGA ACG A; MYH6 F: GCA AAG TAC TGG ATG ACA CGC T; MYH6 R: GTC ATT GCT GAA ACC GAG AAT G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Quorum Spinning Disk Confocal</td>
			<td style="width:203px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">SickKids Imaging Facility</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">ReproCardio hiPS cell derived cardiomyocytes</td>
			<td style="width:203px;">ReproCell</td>
			<td style="width:119px;">RCD001N</td>
			<td style="width:299px;">Positive control for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">RNeasy mini kit</td>
			<td style="width:203px;">Qiagen</td>
			<td style="width:119px;">74106</td>
			<td style="width:299px;">To isolate RNA for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Rotor-Gene SYBR Green PCR Kit</td>
			<td style="width:203px;">Qiagen</td>
			<td style="width:119px;">204074</td>
			<td style="width:299px;">For qPCR with master mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">RPMI 1640</td>
			<td style="width:203px;">Gibco</td>
			<td>A1049101</td>
			<td style="width:299px;">For MSC, monocyte coculture medium.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">TaqMan qPCR primer assays</td>
			<td style="width:203px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">4444556</td>
			<td style="width:299px;">For qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Trypan Blue</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:119px;">T10282</td>
			<td style="width:299px;">Staining of cells for viability and counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Trypsin</td>
			<td style="width:203px;">Gibco</td>
			<td>272500108</td>
			<td style="width:299px;">For cell dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Volocity</td>
			<td style="width:203px;">Perkin-Elmer</td>
			<td style="width:119px;">Volocity 6.3</td>
			<td style="width:299px;">Imaging software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">0.2 &#956;m pore filter</td>
			<td style="width:203px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">566-0020</td>
			<td style="width:299px;">For sterilizing tissue culture media</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:297px;">HERAcell 150i CO2 Incubator</td>
			<td style="width:203px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">51026410</td>
			<td style="width:299px;">For incubating cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Dulbecco&#39;s phosphate buffered saline</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td style="width:299px;">PBS. 1X, Without calcium chloride and magnesium chloride</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Forceps</td>
			<td style="width:203px;">Almedic</td>
			<td style="width:119px;">7727-A10-704</td>
			<td style="width:299px;">For handing rat heart. Can use any similar forceps.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Scissors</td>
			<td style="width:203px;">Fine Science Tools</td>
			<td style="width:119px;">14059-11</td>
			<td style="width:299px;">For mincing rat heart. Curved scissors recommended.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:297px;">50 mL tube</td>
			<td style="width:203px;">BD Falcon</td>
			<td align="right" style="width:119px;">352070</td>
			<td style="width:299px;">For collection during cardiomyocyte collection and general tissue culture procedures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">15 mL tube</td>
			<td style="width:203px;">BD Falcon</td>
			<td align="right" style="width:119px;">352096</td>
			<td style="width:299px;">For general tissue culture procedures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">6-well plates</td>
			<td style="width:203px;">Thermo Scientific Nunc</td>
			<td style="width:119px;">CA73520-906</td>
			<td style="width:299px;">For tissue culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">10 cm tissue culture dishes</td>
			<td style="width:203px;">Corning</td>
			<td style="width:119px;">25382-428</td>
			<td style="width:299px;">For aggregate formation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Axiovert 40C Microscope</td>
			<td style="width:203px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">For bright-field imaging through out tissue culture and the rest of the protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">70 &#956;m cell strainer</td>
			<td style="width:203px;">Fisherbrand</td>
			<td align="right" style="width:119px;">22363548</td>
			<td style="width:299px;">To ensure a single cell suspension before flow cytometry or sorting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Triton X-100</td>
			<td style="width:203px;">EMD Millipore</td>
			<td style="width:119px;">9410-1L</td>
			<td style="width:299px;">Used in permeabilization solution for ICC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Hoechst 33342</td>
			<td style="width:203px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">H1399</td>
			<td style="width:299px;">Stain used during visualization of Cx43 localization</td>
		</tr>
	</tbody>
</table>

in vitro differentiation of human mesenchymal stem cells into functional cardiomyocyte-like cells

Myokardieinfarkt og den efterfølgende iskæmisk cascade resultere i omfattende tab af cardiomyocytes, førende til kongestiv hjerteinsufficiens, den hyppigste årsag til dødelighed på verdensplan. Mesenchymale stamceller (msc) er en lovende mulighed for celle-baserede behandlinger til at erstatte aktuelle, invasive teknikker. MSC'er kan differentiere i mesenchymale lineages, herunder hjerte celletyper, men fuldstændig differentiering af funktionelle celler endnu ikke er nået. Tidligere metoder til differentiering var baseret på farmakologiske midler eller vækstfaktorer. Mere fysiologisk relevante strategier kan imidlertid også aktivere MSCs underkastes cardiomyogenic transformation. Her præsenterer vi en differentiering metode ved hjælp af MSC aggregater på cardiomyocyte feeder lag til at producere cardiomyocyte-lignende ordregivende celler.
Menneskelige navlestrengen perivascular celler (HUCPVCs) har vist sig at have en større differentiering potentielle end almindeligt undersøgt MSC typer, såsom knoglemarv MSCs (BMSCs). Som en ontogenetically yngre kilde undersøgte vi cardiomyogenic potentiale i første trimester (FTM) HUCPVCs i forhold til ældre kilder. FTM HUCPVCs er en roman, rig kilde af MSC'er, der bevarer deres i utero immunoprivileged egenskaber når kulturperler i vitro. Ved hjælp af denne differentiering protokol, FTM og sigt opnået HUCPVCs betydeligt øget cardiomyogenic differentiering i forhold til BMSCs, som angivet ved den øget ekspression af cardiomyocyte markører (dvs. myocyte forstærker faktor 2 C, cardiac troponin T, tunge kæde hjerte myosin, signal regulerende protein α og connexin 43). De bevarede også betydeligt lavere immunogenicitet, som påvist af deres lavere HLA-A udtryk og højere HLA-G udtryk. Anvende aggregat-baserede differentiering, viste FTM HUCPVCs øget samlede dannelse potentielle og genererede kontraherende celler klynger inden for 1 uge i fælles kultur på hjerte feeder lag, bliver den første MSC type hertil.
Vores resultater viser, at denne differentiering strategi effektivt kan udnytte cardiomyogenic potentiale af unge MSC'er, såsom FTM HUCPVCs, og foreslår, at in vitro- pre differentiering kunne være en potentiel strategi til at øge deres regenerativ effekten in vivo.

HUCPVCs Display Higher Aggregate-formation Potential and CD49f Expression Levels Compared to BMSCs:
To induce the differentiation of hMSCs (i.e., FTM HUCPVCs, term HUCPVCs, and BMSCs), single-cell suspensions of undifferentiated MSCs or MSC-containing hanging drops (Table 1) were transferred onto rat primary cardiomyocyte monolayers to establish direct co-cultures or aggregate co-cultur...

Watch this Scientific Journal Video about In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells at JoVE.com

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Vi præsenterer her, en metode til at effektivt udnytte hjerte differentiering potentiale af unge kilder til humane mesenkymale stamceller for at skabe funktionelle, ordregivende, cardiomyocyte-lignende celler in vitro-.

In Vitro Differentiering af humane mesenkymale stamceller i funktionel Cardiomyocyte-lignende celler

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Research

JoVE Journal

Developmental Biology

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Instrueret differentiering af Primitive og endelige hæmatopoietisk ophav fra humane pluripotente stamceller

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In vitro differentiering af humane pluripotente stamceller til trofoblastceller

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Horisontal Hel Mount: En Novel Processing and Imaging Protocol for Tykke, Tredimensionale Tissue Tværsnit af Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Hurtig, instrueret differentiering af retinale Pigment epitel celler fra menneskelige embryonale eller induceret pluripotente stamceller

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Encellede Photoconversion i levende intakt zebrafisk

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Beregningsmæssige analyse af Caenorhabditis elegans kønscelleoverførsel at studere Distribution kerner, proteiner og cytoskelettet

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Afledning og differentiering af Canine æggestokkene mesenkymale stamceller

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Effektiv differentiering af humane pluripotente stamceller i leverceller

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

I Vitro Generation af Somite derivater fra menneskelige induceret celler pluripotente stamceller

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Rettet differentiering af hæmogene endotelceller fra humane pluripotente stamceller

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...