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

著者らは彼のプラスミドについてXiaobin Zhangに感謝したい。私たちはまた、実験中に彼らの親切な助けをしてくれたShaorong GaoとQianfei Wangにも感謝します。この研究は、中国国立自然科学財団（No.81101346,81271963,81100331）、北京215高水準人材養成プロジェクト（No.2014-3-025）、北京チャオヤン病院基金（No CYXX-2017-01）、中国科学アカデミー青年イノベーション推進協会（YL）

PBCsからhiPSCを生成するためのプロトコールは、我々の以前の研究5に見出すことができる。この研究は、私たちの機関の機関審査委員会によって承認されました。 1.胚様体（EB）形成<ol><li> 5％ウシ胎児血清（FBS）、1×非必須アミノ酸、55μM2-メルカプトエタノール、2mM L-メルカプトエタノール、2mM L-メチオニン、および2mM LPSを補充したノックアウトダ?...

<ol>
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H., 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=Generation+of+induced+pluripotent+stem+cells+from+human+blood.">Generation of induced pluripotent stem cells from human blood.</a> Blood. 113 (22), 5476-5479 (2009).</li><li>Zhang, X. 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. A., Penick, K. J., Welter, J. F. <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+bone+marrow-derived+mesenchymal+stem+cells:+tips+and+tricks.">Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells: tips and tricks.</a> Methods Mol Biol. 698, 253-278 (2011).</li><li>Oldershaw, R. A., 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=Directed+differentiation+of+human+embryonic+stem+cells+toward+chondrocytes.">Directed differentiation of human embryonic stem cells toward chondrocytes.</a> Nat Biotechnol. 28 (11), 1187-1194 (2010).</li><li>Monje, L., Varayoud, J., Luque, E. H., Ramos, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+exposure+to+bisphenol+A+modifies+the+abundance+of+estrogen+receptor+alpha+transcripts+with+alternative+5'-untranslated+regions+in+the+female+rat+preoptic+area.">Neonatal exposure to bisphenol A modifies the abundance of estrogen receptor alpha transcripts with alternative 5'-untranslated regions in the female rat preoptic area.</a> J Endocrinol. 194 (1), 201-212 (2007).</li><li>Teramura, T., 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=Induction+of+mesenchymal+progenitor+cells+with+chondrogenic+property+from+mouse-induced+pluripotent+stem+cells.">Induction of mesenchymal progenitor cells with chondrogenic property from mouse-induced pluripotent stem cells.</a> Cell Reprogram. 12 (3), 249-261 (2010).</li><li>Su, R. 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. Y., 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=Efficient+differentiation+of+human+iPSC-derived+mesenchymal+stem+cells+to+chondroprogenitor+cells.">Efficient differentiation of human iPSC-derived mesenchymal stem cells to chondroprogenitor cells.</a> J Cell Biochem. 114 (2), 480-490 (2013).</li><li>Koyama, N., 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=Human+induced+pluripotent+stem+cells+differentiated+into+chondrogenic+lineage+via+generation+of+mesenchymal+progenitor+cells.">Human induced pluripotent stem cells differentiated into chondrogenic lineage via generation of mesenchymal progenitor cells.</a> Stem Cells Dev. 22 (1), 102-113 (2013).</li><li>Liu, X., 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=Role+of+insulin-transferrin-selenium+in+auricular+chondrocyte+proliferation+and+engineered+cartilage+formation+in+vitro.">Role of insulin-transferrin-selenium in auricular chondrocyte proliferation and engineered cartilage formation in vitro.</a> Int J Mol Sci. 15 (1), 1525-1537 (2014).</li><li>Guzzo, R. 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>

ここでは、iPSCを介してPBCから軟骨細胞を生成するためのプロトコールを提供する。 PBCはより一般的であり、臨床分野で広く使用されているので、それらは再プログラムのための潜在的な代替物として提示される。この研究では、エピソームベクター（EV）を用いて、Zhang らによって確立された方法に従って、PBCをiPSCに再プログラムした。 11 。この統合のない?...

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

軟骨組織は、自己修復および再生の能力が非常に低い。様々な外科的介入および生物学的処置が、不十分な結果を伴って軟骨および関節機能を回復させるために使用される。最近の幹細胞技術の発展は、軟骨修復領域全体を変化させる可能性がある1 。様々な幹細胞が種細胞として研究されてきたが、拒絶反応を起こさずに多くのタイプの患者特異的細胞を提供することができるため、ヒト誘導性多能性幹細胞（hiPSC）が最も有望な選択肢であるようである2 。さらに、成人細胞の限られた増殖性を克服し、自己複製能および多能性を維持することができる。さらに、特定のタイプの軟骨細胞を得るために遺伝子ターゲティングを用いて遺伝子型を変えることができる。 再プログラミングの可能性も十分に研究されているので、繊維芽細胞はiPSCを生成するために広く使用されている。しかし、患者からの痛みを伴う生検や、線維芽細胞のin vitroでの拡大が必要であり、遺伝子突然変異を引き起こす可能性があるなど、まだ克服しなければならないいくつかの限界が存在する3 。最近、PBCはリプログラミング4にとって有利であることが判明した。さらに、それらは一般に利用され豊富に貯蔵されていた。研究の焦点を皮膚からリダイレクトする可能性があります。しかし、私たちが知る限りでは、軟骨細胞への分化が続くPBCの再プログラミングに関する報告はほとんどありません。 現在の研究では、軟骨細胞の形成を模倣するために、iPSCをそれらをiPSCに再プログラミングし、次いでペレット培養系を介して軟骨系統に分化させることによって、PBCを代替源として利用する。

<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

この研究では、統合された方法で誘導された多能性幹細胞（iPSCs）を介して軟骨細胞を産生するために、末梢血細胞（PBC）を種細胞として使用した。胚様体（EB）形成および線維芽細胞増殖に続いて、iPSCは、無血清および無細胞条件下で21日間軟骨分化のために誘導される。軟骨細胞誘導後、形態学的、免疫組織化学的および生化学的分析、ならびに軟骨形成性分化マーカーの定量的リアルタイムPCR検査によって、細胞の表現型を評価する。軟骨形成性ペレットは、アルシアンブルーおよびトルイジンブルー染色が陽性であることを示す。コラーゲンIIおよびX染色の免疫組織化学も陽性である。硫酸化グリコサミノグリカン（sGAG）含量および軟骨分化マーカーCOLLAGEN2 （ COL2 ）、 COLLAGEN10 （ COL10 ）、 SOX9およびAGGRECANは、hiPSCsおよび線維芽細胞と比較して非還元性のペレットが得られた。これらの結果は、PBCを軟骨修復のためのiPSCsを生成するための種細胞として使用することができることを示唆しており、これは患者特異的かつ費用対効果が高い。

hiPSCsの軟骨形成分化： EB形成培地および基礎培地を使用してhiPSCを間葉系統に分化させた。多段階培養法を用いた（ 図1 ）。最初に、hiPSCを10日間EB形成により自発的に分化させた（D10; 図2A ）。第2に、細胞はEBからさらに10日間（D10 + 10）逸脱した。これらの2つのステップの間、i...

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

我々は、胚様体（EB）形成、線維芽細胞増殖および軟骨形成誘導を含む無組み込み法を用いて、誘導末梢血（PB）から誘導された多能性幹細胞（iPSC）を介して軟骨系統を生成するためのプロトコールを提示する。

末梢血由来ヒト誘導多能性幹細胞からの軟骨細胞の分化

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

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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

エピソームベクトルを用いたヒト末梢血単核細胞からの統合フリー人工多能性幹細胞の生成

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

臍帯血由来誘導多能性幹細胞からの軟骨形成ペレット形成

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

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

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

臍帯血由来から 3 D 肌 Organoid の生成誘導多能性幹細胞

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

体節誘導体誘導される人間からの体外世代多能性幹細胞します。

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

ヒト人工多能性幹細胞から誘導されるインビトロ神経筋接合

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

ヒト多能性幹細胞からの血原性内皮細胞の指向性分化

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