Name: differentiating chondrocytes from peripheral blood-derived human induced pluripotent stem cells
Uploaded: 2017-07-18T14:00:00
Description: Watch this Scientific Journal Video about Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells at JoVE.com

Författarna vill tacka Xiaobin Zhang för sin plasmid. Vi tackar också Shaorong Gao och Qianfei Wang för deras vänliga hjälp under experimentet. Denna studie stöds av National Natural Science Foundation of China (No.81101346, 81271963, 81100331), Peking 215 högtalareprojekt (nr.2014-3-025) och Peking Chao-Yang sjukhusfonden (nr . CYXX-2017-01) och Youth Innovation Promotion Association av kinesiska vetenskapsakademin (YL).

Protokollet för generering av hiPSC från PBC kan hittas i vår tidigare studie 5 . Studien godkändes av institutionens institutionella granskningsråd. 1. Embryoid Body (EB) Formation <ol><li> Gör 50 ml hiPSC-medium: Knockout Dulbeccos Modified Eagle Medium (DMEM) kompletterat med 15% knockout serumutbyte (KSR), 5% fetalt bovint serum (FBS), 1 x icke-essentiella aminosyror, 55 | iM 2-merkaptoetanol, 2 mM L -glutamin och 8 ng / ml basisk fibroblasttillväxtfaktor...

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+reprogramming+of+human+peripheral+blood+cells.">Cellular reprogramming of human peripheral blood cells.</a> Genomics Proteomics Bioinformatics. 11 (5), 264-274 (2013).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+blood+cells+into+induced+pluripotent+stem+cells+as+a+new+cell+source+for+cartilage+repair.">Reprogramming of blood cells into induced pluripotent stem cells as a new cell source for cartilage repair.</a> Stem Cell Res Ther. 7 (31), (2016).</li><li>Canene-Adams, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+formalin-fixed+paraffin-embedded+tissue+for+immunohistochemistry.">Preparation of formalin-fixed paraffin-embedded tissue for immunohistochemistry.</a> Methods Enzymol. 533, 225-233 (2013).</li><li>Solchaga, L. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+integration-free+ips+cells+from+human+adult+peripheral+blood+using+BCL-XL+together+with+Yamanaka+factors.">Efficient generation of integration-free ips cells from human adult peripheral blood using BCL-XL together with Yamanaka factors.</a> PLoS One. 8 (5), e64496 (2013).</li><li>Carey, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+factor+stoichiometry+influences+the+epigenetic+state+and+biological+properties+of+induced+pluripotent+stem+cells.">Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells.</a> Cell Stem Cell. 9 (6), 588-598 (2011).</li><li>Okita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+nonviral+method+to+generate+integration-free+human-induced+pluripotent+stem+cells+from+cord+blood+and+peripheral+blood+cells.">An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.</a> Stem Cells. 31 (3), 458-466 (2013).</li><li>Staerk, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+peripheral+blood+cells+to+induced+pluripotent+stem+cells.">Reprogramming of human peripheral blood cells to induced pluripotent stem cells.</a> Cell Stem Cell. 7 (1), 20-24 (2010).</li><li>Qu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+differentiation+of+human+pluripotent+stem+cells+in+chondrocyte+co-culture.">Chondrogenic differentiation of human pluripotent stem cells in chondrocyte co-culture.</a> Int J Biochem Cell Biol. 45 (8), 1802-1812 (2013).</li><li>Guzzo, R. M., Gibson, J., Xu, R. H., Lee, F. 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M., Scanlon, V., Sanjay, A., Xu, R. H., Drissi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+cell+type-specific+iPS+cells+with+enhanced+chondrogenic+potential.">Establishment of human cell type-specific iPS cells with enhanced chondrogenic potential.</a> Stem Cell Rev. 10 (6), 820-829 (2014).</li><li>Goepfert, C., Slobodianski, A., Schilling, A. F., Adamietz, P., Portner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+engineering+from+mesenchymal+stem+cells.">Cartilage engineering from mesenchymal stem cells.</a> Adv Biochem Eng Biotechnol. 123, 163-200 (2010).</li><li>Ingber, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+and+developmental+biology:+going+biomimetic.">Tissue engineering and developmental biology: going biomimetic.</a> Tissue Eng. 12 (12), 3265-3283 (2006).</li><li>Yoshida, Y., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+stem+cell+advances:+induced+pluripotent+stem+cells+for+disease+modeling+and+stem+cell-based+regeneration.">Recent stem cell advances: induced pluripotent stem cells for disease modeling and stem cell-based regeneration.</a> Circulation. 122 (1), 80-87 (2010).</li></ol>

Här tillhandahåller vi ett protokoll för att generera kondrocyter från PBC via iPSCs. Eftersom PBC är vanligare och används i stort på kliniska fält presenteras de som ett potentiellt alternativ för omprogrammering. I denna studie användes episomala vektorer (EV) för att omprogrammera PBC till iPSCs, enligt metoden som fastställdes av Zhang et al. 11 . Detta integrationsfria tillvägagångssätt involverar inte integrering av virusassocierad genotoxicitet, vilket antas ha en ...

An erratum was issued for: <a href="https://www.jove.com/video/55722/differentiating-chondrocytes-from-peripheral-blood-derived-human">Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells</a>.
The corresponding author was updated from:
Yueying Li 
yueyinglee2010@hotmail.com 
Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences
The corresponding author is now listed as:
Tie Liu 
waterkins@163.com 
Department of Orthopedics, Beijing Chao-Yang Hospital, Capital Medical University

An erratum was issued for: <a href="https://www.jove.com/video/55722/differentiating-chondrocytes-from-peripheral-blood-derived-human">Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells</a>.

Erratum: Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

Bruskvävnad har en mycket dålig kapacitet för självreparation och regenerering. Olika kirurgiska ingrepp och biologiska behandlingar används för att återställa brusk och ledfunktion, med otillfredsställande resultat. Den senaste utvecklingen av stamcellsteknologin kan ändra hela bruskreparationsfältet 1 . Olika stamceller har studerats som fröceller, men humana inducerade pluripotenta stamceller (hiPSCs) verkar vara det mest lovande valet, eftersom de kan ge många typer av patientspecifika celler utan att orsaka avstötningsreaktioner 2 . Dessutom kan de övervinna den vuxna cellens begränsade proliferativa natur och behålla sin självförnyelse och pluripotenta förmågor. Dessutom kan genriktning användas för att ändra genotypen för att erhålla specifika typer av kondrocyter. Fibroblaster har använts i stor utsträckning för att generera iPSCs eftersom deras omprogrammeringspotentialer också har blivit väl studerade.Det finns emellertid fortfarande vissa begränsningar som måste övervinnas, såsom den smärtsamma biopsin från patienter och behovet av in vitro- expansion av fibroblasterna, vilket kan resultera i genmutationer 3 . Nyligen visade sig PBCs vara fördelaktiga för omprogrammering 4 ; Dessutom användes de vanligen och lagras rikligt. Det är möjligt att de kan omdirigera studiefokus från huden. Men, så vitt vi vet, finns det få rapporter om PBC-omprogrammering följt av differentiering till kondrocyter. I den aktuella studien använder vi PBC som en alternativ källa genom att omprogrammera dem till iPSCs och sedan differentiera iPSC: erna i kondrogena linjen genom ett pelletsodlingssystem för att efterlikna kondrocytbildning.

<table>
	<tbody>
		<tr>
			<td>Knockout DMEM</td>
			<td>Invitrogen</td>
			<td>10829018</td>
			<td>Basal medium used for hiPSC culture and EB formation medium</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KSR)</td>
			<td>Invitrogen</td>
			<td>10828028</td>
			<td>A more defined, FBS-free medium supplement used for hiPSC culture and EB formation medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>sh30070.03</td>
			<td>Used for hiPSC culture and EB formation medium,offers excellent value for cell culture</td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>Chemicon</td>
			<td>TMS-001-C</td>
			<td>Used as a growth supplement in all the cell culture medium, to increase cell growth and viability</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>An amino acid required for cell culture</td>
		</tr>
		<tr>
			<td>Basic fibroblast growth factor (bFGF)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>A cytokine used for sustaining the pluripotency and self-renewal of hiPSCs</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Invitrogen</td>
			<td>17105041</td>
			<td>Used for hiPSC dissociation for subculture</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>C11960</td>
			<td>Basal medium used for MSC culture medium</td>
		</tr>
		<tr>
			<td>0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
			<td>Used for cell attachment onto the dishes</td>
		</tr>
		<tr>
			<td>0.25% trypsin/EDTA</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td>Used for cell dissociation</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td>A balanced salt solution used for cell wash or reagent preparing</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>invitrogen</td>
			<td>21985023</td>
			<td>Used as a growth supplement in all the cell culture medium.</td>
		</tr>
		<tr>
			<td>ITS</td>
			<td>invitrogen</td>
			<td>41400045</td>
			<td>Insulin, Transferrin, Selenium Solution.Used for chondrogenic differentiation.</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>4403</td>
			<td>Known as vitamin C. It helps in active growth and has antioxidant property.</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td>Added to cell culture medium as an energy source in addition to glucose.</td>
		</tr>
		<tr>
			<td>Transforming growth factor-beta 1</td>
			<td>Peprotech</td>
			<td>AF-100-21C</td>
			<td>A cytokine that regulate cell proliferation, growth and chondrogenic differentiation.</td>
		</tr>
		<tr>
			<td>Rabbit polyclonal antibodies against Collagen II</td>
			<td>Abcam</td>
			<td>ab34712</td>
			<td>This antibody reacts with Type II collagens,which is specific for cartilaginous tissues.</td>
		</tr>
		<tr>
			<td>Mouse monoclonal antibodies to Collagen X</td>
			<td>Abcam</td>
			<td>ab49945</td>
			<td>This antibody reacts with Type X collagen,which is a product of hyperthrophic chondrotocytes.</td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td>For mounting and long-term storage of slides</td>
		</tr>
		<tr>
			<td>Toluidine blue</td>
			<td>Sigma</td>
			<td>89640</td>
			<td>Used for proteoglycans detection.</td>
		</tr>
		<tr>
			<td>Alcian blue</td>
			<td>Amresco</td>
			<td>#0298</td>
			<td>Used for glucosaminoglycans detection.</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma</td>
			<td>P4762-25MG</td>
			<td>Used to digest chondrogenic pellets.</td>
		</tr>
		<tr>
			<td>Dimethylmethylene blue</td>
			<td>Sigma</td>
			<td>341088-1G</td>
			<td>Used to quantitate glycosaminoglyans</td>
		</tr>
		<tr>
			<td>Chondroitin sulfate sodium salt from shark cartilage</td>
			<td>Sigma</td>
			<td>C4384-250MG</td>
			<td>Used to draw the standard curve for sGAG content measurement.</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS assay kit</td>
			<td>Invitrogen</td>
			<td>Q32851 (100)</td>
			<td>Used to determine DNA content</td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>Used for RNA isolation from cells</td>
		</tr>
		<tr>
			<td>Reverse Transcriptase System</td>
			<td>Promega</td>
			<td>A3500</td>
			<td>Used to convert RNA into cDNA</td>
		</tr>
		<tr>
			<td>SYBR FAST qPCR kit Master Mix</td>
			<td>Kapa</td>
			<td>KK4601</td>
			<td>Used for Real-time PCR</td>
		</tr>
	</tbody>
</table>

differentiating chondrocytes from peripheral blood-derived human induced pluripotent stem cells

I denna studie använde vi perifera blodceller (PBC) som fröceller för att producera kondrocyter via inducerade pluripotenta stamceller (iPSCs) i en integrationsfri metod. Efter embryoidkropps (EB) -bildning och fibroblastisk cellexpansion induceras iPSCs för kondrogenisk differentiering under 21 dagar under serumfria och xenofria betingelser. Efter kondrocytinduktion utvärderas fenotyperna hos cellerna genom morfologiska, immunohistokemiska och biokemiska analyser, liksom av den kvantitativa realtids PCR-undersökningen av kondrogena differentieringsmarkörer. Kondrogena pellets visar positiv alcianblå och toluidinblåfärgning. Immunohistokemin för kollagen II och X-färgning är också positiv. Det sulfaterade glykosaminoglykan-innehållet (sGAG) och de kondrogena differentieringsmarkörerna COLLAGEN 2 ( COL2 ), COLLAGEN 10 ( COL10 ), SOX9 och AGGRECAN är signifikant uppreglerade i tjondRogeniska pellets jämfört med hiPSC och fibroblastiska celler. Dessa resultat tyder på att PBC kan användas som fröceller för att generera iPSCs för broskreparation, vilket är patientspecifikt och kostnadseffektivt.

Kondrogen Differensiering av hiPSC: er: EB-bildningsmedium och basal odlingsmedium användes för att differentiera hiPSC: erna i mesenkymstammen. En kultur med flera steg användes ( figur 1 ). Först differentierades hiPSC: erna spontant via EB-bildning under 10 dagar (D10; Figur 2A ). För det andra utbröt cellerna från EB i ytterligare 10 dagar (D10 + 10). Under de...

Watch this Scientific Journal Video about Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells at JoVE.com

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

Vi presenterar ett protokoll för att generera en kondrogenstamning från humant perifert blod (PB) via inducerade pluripotenta stamceller (iPSCs) med hjälp av en integrationsfri metod som innefattar embryoidkropps (EB) -bildning, expansion av fibroblastiska celler och kondrogeninduktion.

Differentierande kondrocyter från perifert blod härledda humana inducerade plöripotenta stamceller

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Research

JoVE Journal

Developmental Biology

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

Tredimensionell Kvantifiering av Dendritutskotten från Pyramidal nervceller Härstammar från Human inducerade pluripotenta stamceller

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

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Generering av inducerade pluripotenta stamceller från humana perifera T-celler med Sendai virus i Feeder fria förhållanden

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Generering av inducerade pluripotenta stamceller från humant melanom tumörinfiltrerande lymfocyter

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Generation Integrations-fria inducerade pluripotenta stamceller från humant perifert blod mononukleära celler med användning av episomala vektorer

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Kondrogen pelletsbildning från Cord Blood-härledda inducerade Pluripotenta stamceller

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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

Snabb, regisserad differentiering av Retinal Pigment epitelial celler från mänskliga embryonala eller inducerade pluripotenta stamceller

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

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Generation av 3D hud Organoid från sladd blod-derived inducerade pluripotenta stamceller

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

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

I Vitro Generation av Somite derivat från mänskliga inducerade stamceller pluripotenta

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

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

In vitro Neuromuskulära Junction inducerad från humana inducerade pluripotenta stamceller

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Riktad differentiering av hemogena endotelceller från humana pluripotenta stamceller

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