Differentiering og karakterisering af osteoklaster fra humant inducerede pluripotente stamceller
Summary
Denne protokol præsenterer differentieringen af humane osteoklaster fra inducerede pluripotente stamceller (iPSC’er) og beskriver metoder til karakterisering af osteoklaster og osteoklastprækursorer.
Abstract
Denne protokol beskriver udbredelsen og overførslen af humane iPSC’er og deres differentiering i osteoklaster. For det første dissocieres iPSC’er i en enkeltcellesuspension til videre anvendelse i embryoid kropsinduktion. Efter mesodermal induktion gennemgår embryoidlegemer hæmatopoietisk differentiering, hvilket producerer en flydende hæmatopoietisk cellepopulation. Derefter gennemgår de høstede hæmatopoietiske celler et makrofagkolonistimulerende faktormodningstrin og endelig osteoklastdifferentiering. Efter osteoklastdifferentiering er osteoklaster karakteriseret ved farvning til TRAP i forbindelse med en methylgrøn nuklear plet. Osteoklaster observeres som multinucleated, TRAP + polykaryoner. Deres identifikation kan understøttes yderligere af Cathepsin K-farvning. Knogler og mineraleresorptionsassays muliggør funktionel karakterisering, hvilket bekræfter identiteten af bona fide osteoklaster. Denne protokol demonstrerer en robust og alsidig metode til at differentiere humane osteoklaster fra iPSC’er og giver mulighed for nem anvendelse i applikationer, der kræver store mængder funktionelle humane osteoklaster. Anvendelser inden for knogleforskning, kræftforskning, vævsteknik og endoprosteseforskning kunne forestilles.
Introduction
Osteoklaster (OC’er) er hæmatopoietisk afledte 1,2, alsidige celletyper, der almindeligvis anvendes af forskere inden for områder som knoglesygdomsforskning 3,4, kræftforskning 5,6, vævsteknik 7,8 og endoprosteseforskning 9,10. Ikke desto mindre kan OC-differentiering være udfordrende, da fusion af mononukleære prækursorer til multinucleated OC’er er nødvendig for at skabe funktionelle OC’er11. Flere biologiske faktorer, såsom receptoraktivator af NF-κB ligand (RANKL) og makrofagkolonistimulerende faktor (M-CSF), er nødvendige for OC-differentiering. M-CSF er blevet rapporteret at have en positiv effekt på celleproliferation, celleoverlevelse og RANK-ekspression 12,13,14. På den anden side binder RANKL til RANK, som aktiverer nedstrøms signalkaskader, der inducerer osteoklastogenese. Aktivering medieres via TNF-receptorassocieret faktor 6 (TRAF6), hvilket fører til nedbrydning af nuklear faktor af kappa light polypeptid gene enhancer i B-celleinhibitor, alfa (IκB-α), et bindende protein, der binder NF-kB dimerer16,17. Derfor frigiver IκB-α nedbrydning NF-kB-dimerer, som derefter translokerer ind i kernen og inducerer ekspressionen af transkriptionsfaktorerne c-Fos og nuklear faktor for aktiverede T-celler 1 (NFATc1). Dette udløser igen transkriptionen af en lang række OC-differentieringsrelaterede proteiner15,18. Opregulerede proteiner såsom DC-stempel og Atp6v0d2 medierer celle-cellefusion af OC-prækursorer, hvilket fører til syncytiumdannelse 19,20,21.
Med hensyn til humane primære celler er CD34+ og CD14+ PBMC’er i øjeblikket de mest anvendte celletyper til differentiering i OC’er22. Denne tilgang er imidlertid begrænset af heterogeniteten inden for CD34+ -populationen af høstede celler fra donorer23 og deres begrænsede udvidelsesmuligheder. Menneskelige iPSC’er udgør en alternativ kilde til OC’er. Da de kan formeres på ubestemt tid24, giver de mulighed for udvidelse og opskalering af OC-produktion. Dette giver mulighed for differentiering af et stort antal OC’er, hvilket letter OC-forskning.
Flere protokoller til differentiering af iPSC’er i OC’er er blevet offentliggjort 25,26,27. Hele differentieringsprocessen kan opdeles i en iPSC-formeringsdel, en mesodermal og hæmatopoietisk differentieringsdel og OC-differentiering. Formering af iPSC’er før differentieringsprocessen giver mulighed for opskalering af OC-produktion inden differentiering. Der findes flere tilgange til mesodermal og hæmatopoietisk differentiering. Traditionelt er embryoid kropsdannelse (EB) blevet brugt til at differentiere hæmatopoietiske celler, men monolagsbaserede tilgange repræsenterer en anden hæmatopoietisk differentieringsstrategi, der ikke kræver EB-induktion. Ikke desto mindre synes enkeltlagsbaserede systemer at kræve yderligere optimering, da vi og andre har fundet EB-baserede tilgange til at være mere robuste til differentiering af OC’er.
Her beskriver vi differentieringen af OC’er fra humane iPSC’er ved hjælp af en EB-baseret protokol. Denne protokol blev tilpasset fra Rössler et al.26 og modificeret for at øge robustheden og muliggøre kryopræservering under differentieringsprocessen. For det første høstede vi hæmatopoietiske celler kun én gang efter 10 dages differentiering. Hæmatopoietiske celler blev derefter kryopræserveret for at give mulighed for mere fleksibilitet under differentieringsprocessen. Derudover øgede vi den hæmatopoietiske cellesåtæthed fra 1 x 105 til 2 x 105 celler / cm2 for OC-differentiering. Et nyere humant iPSC-serumfrit medium (hiPSC-SFM, se materialetabel) blev anvendt, og belægning af brønde blev udført med 200-300 μg/ml basalmembranekstrakt (se materialetabel) i stedet for 0,1% gelatine. Penicillin/streptomycin blev ikke tilsat medierne.
Protokollen af Rössler et al.26 blev oprindeligt tilpasset fra en iPSC til en makrofagdifferentieringsprotokol28, der bruger EB-dannelse til hæmatopoietisk differentiering. Mens EB-dannelse er blevet brugt i længere tid af forskere til hæmatopoietisk differentiering29,30, er flere metoder til EB-induktion blevet beskrevet i litteraturen, såsom spontan aggregering, centrifugering i en rundbundet brøndplade, hængende dråbekultur, bioreaktorkultur, konisk rørkultur, langsomt drejende lateral beholder og mikroskimmelgelkultur31. Denne protokol anvender centrifugering af dissocierede iPSC’er i en rundbundet brøndplade for at bringe enkelte iPSC-celler i nærheden af hinanden og for at muliggøre kugledannelse (EB) som beskrevet nedenfor.
Protocol
Representative Results
Discussion
Denne protokol tilbyder en pålidelig og robust metode til at differentiere iPSC’er i OC’er. Ikke desto mindre er der flere faldgruber, der kan opstå gennem differentieringsprocessen. Humane iPSC-linjer genereret fra celler af forskellig vævsoprindelse er med succes blevet differentieret ved hjælp af denne protokol33. Ved frysning af iPSC’er (se protokoltrin “3. Frysning tilbage iPSC’er”), blev en brønd på tidspunktet for passaging frosset tilbage til en kryoial. Ved optøning (se protokoltri…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne takke medlemmerne af Giachelli-laboratoriet for deres tekniske hjælp og støtte. Vi takker W. M. Keck Microscopy Center og Keck Center manager, Dr. Nathanial Peters, for hjælp med at opnå konfokal mikroskopi og widefield mikroskopi billeder. Vi takker også UW Flow Core Facility og Flow Core Facility manager, Aurelio Silvestroni, for teknisk support og assistance. Endelig takker vi Hannah Blümke for støtten med illustration og grafisk design.
Finansieringen blev ydet gennem National Institutes of Health-tilskud R35 HL139602-01. Vi anerkender også NIH S10-tilskud S10-OD016240 til instrumentfinansiering på W. M. Keck Center samt NIH-tilskud 1S10OD024979-01A1 til instrumentfinansiering på UW Flow Core Facility.
Materials
| 2-Mercaptoethanol | Sigma Aldrich | M6250-10ML | |
| Antibody – Anti-Cathepsin K | Abcam | ab19027 | |
| Antibody – APC-conjugated Anti-Human CD45 | BD | 555485 | |
| Antibody – APC-conjugated Mouse IgG1, κ Isotype Control | BD | 555751 | |
| Antibody – BV711-conjugated Anti-Human CD14 | BD | 563372 | |
| Antibody – BV711-conjugates Mouse IgG2b, κ Isotype Control | BD | 563125 | |
| Antibody – Goat Anti-Rabbit IgG H&L Alexa Fluor® 647 | Abcam | ab150079 | |
| Antibody – PE-conjugated Anti-Human CD14 | R&D Systems | FAB3832P-025 | |
| Antibody – PE-conjugated Anti-Human Integrin alpha M/CD11b | R&D Systems | FAB16991P-025 | |
| Antibody – PE-Cy7-conjugated Anti-Human CD34 | BD | 560710 | |
| Antibody – PE-Cy7-conjugated Mouse IgG1 κ Isotype Control | BD | 557872 | |
| Antibody – PE/Cyanine5-conjugated Anti-Human CD11b | Biolegend | 301308 | |
| Antibody – PE/Cyanine5-conjugated Mouse IgG1, κ Isotype Ctrl | Biolegend | 400118 | |
| Antibody – PerCP-Cy5.5-conjugated Mouse IgG1 κ Isotype Control | BD | 550795 | |
| Antibody – PerCpCy5.5-conjugated Anti-Human CD43 | BD | 563521 | |
| Bone Resorption Assay Kit | CosmoBioUSA | CSR-BRA-24KIT | |
| Countess 3 Automated Cell Counter | ThermoFisher | 16812556 | |
| Cultrex Stem Cell Qualified Reduced Growth Factor Basement Membrane Extract | R&D Sytems | 3434-010-02 | Basal membrane extract |
| DAPI | R&D Systems | 5748/10 | |
| Dispase (5 U/mL) | STEMCELL Technologies | 7913 | |
| DMEM/F-12 with 15 mM HEPES | Stem Cell | 36254 | |
| DMSO | Sigma Aldrich | D2650 | |
| DPBS | Sigma Aldrich | D8537-500ML | |
| Human Bone Morphogenetic Protein 4 (hBMP4) | STEMCELL Technologies | 78211 | |
| Human IL-3 | STEMCELL Technologies | 78146.1 | |
| Human Macrophage Colony-stimulating Factor (hM-CSF) | STEMCELL Technologies | 78150.1 | |
| Human Soluble Receptor Activator of Nuclear Factor-κB Ligand (hsRANKL) | STEMCELL Technologies | 78214.1 | |
| Human Stem Cell Factor (hSCF) | STEMCELL Technologies | 78155.1 | |
| Human TruStain FcX (Fc Receptor Blocking Solution) | Biolegend | 422301 | |
| Human Vascular Endothelial Growth Factor-165 (hVEGF165) | STEMCELL Technologies | 78073 | |
| Invitrogen Rhodamine Phalloidin | Invitrogen | R415 | |
| MEM α, nucleosides, no phenol red | ThermoFisher | 41061029 | |
| mFreSR | STEMCELL Technologies | 05855 | Serum free cryopreservation medium |
| mTeSR Plus medium | STEMCELL Technologies | 100-0276 | Human iPSC-serum free medium (hiPSC-SFM) |
| Nunclon Sphera 96-Well, Nunclon Sphera-Treated, U-Shaped-Bottom Microplate | Thermo Scientific | 174925 | Round bottom ultra-low attachment 96-well plate |
| P1000 Wide Bore Tips | ThermoFisher | 2079GPK | |
| ROCK-Inhibitor Y-27632 | STEMCELL Technologies | 72304 | |
| StemSpan SFEM | StemCell | 09650 | Hematopoietic cell culture medium |
| TrypLE Select Enzyme (1X), no phenol red | Thermo Fisher | 12563011 | Single-cell dissociation reagent |
| Ultraglutamine | Bioscience Lonza | BE17-605E/U1 | |
| X-VIVO 15 Serum-free Hematopoietic Cell Medium | Bioscience Lonza | 04-418Q | Hematopoietic basal medium |
| µ-Slide 8 Well High | Ibidi | 80806 |
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