The authors wish to thank Xiaobin Zhang for his plasmid. We also thank Shaorong Gao and Qianfei Wang for their kind help during the experiment. This study is supported by the National Natural Science Foundation of China (No.81101346, 81271963, 81100331), the Beijing 215 high-level talent project (No.2014-3-025), and the Beijing Chao-Yang Hospital Fund (No. CYXX-2017-01), and the Youth Innovation Promotion Association of Chinese Academy of Sciences (Y.L.).

The protocol for the generation of hiPSCs from PBCs can be found in our previous study5. The study was approved by the Institutional Review Board of our institution.
1. Embryoid Body (EB) Formation
<ol>
	<li>Make 50 mL of hiPSC medium: Knockout Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 15% knockout serum replacement (KSR), 5% fetal bovine serum (FBS), 1&#215; nonessential amino acids, 55 &#956;M 2-mercaptoethanol, 2 mM L-glutamine, and 8 ng/mL basi...

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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. 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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. 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>

Here, we provide a protocol to generate chondrocytes from PBCs via iPSCs. Because PBCs are more common and widely used in the clinical field, they are presented as a potential alternative for reprogramming. In this study, episomal vectors (EV) were utilized to reprogram PBCs into iPSCs, following the method established by Zhang et al.11. This integration-free approach does not involve integrating virus-associated genotoxicity, which is believed to have a broad effect in the clinical field...

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

Cartilage tissue has a very poor capacity for self-repair and regeneration. Various surgical interventions and biological treatments are used to restore cartilage and joint function, with unsatisfying results. The recent development of stem cell technology may change the entire cartilage repair field1. Various stem cells have been studied as seed cells, but human induced pluripotent stem cells (hiPSCs) appear to be the most promising choice, as they can provide many types of patient-specific cells without causing rejection reactions2. Furthermore, they can overcome the limited proliferative nature of adult cells and maintain their self-renewal and pluripotent abilities. Moreover, gene targeting can be used to change the genotype to obtain specific types of chondrocytes.
Fibroblasts have been widely used to generate iPSCs because their reprogramming potentials have also been well studied. However, there are still some limitations that must be overcome, such as the painful biopsy from patients and the need for the in vitro expansion of the fibroblasts, which may result in gene mutations3. Recently, PBCs were found to be advantageous for reprogramming4; moreover, they were commonly utilized and abundantly stored. It is possible that they may redirect study focus from the skin. However, to the best of our knowledge, there are few reports on PBC reprogramming followed by differentiation into chondrocytes.
In the current study, we utilize PBCs as an alternative source by reprogramming them into iPSCs and then differentiating the iPSCs into the chondrogenic lineage through a pellet culture system in order to mimic chondrocyte formation.

<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

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

Chondrogenic Differentiation of hiPSCs:
EB formation medium and basal culture medium were used to differentiate the hiPSCs into the mesenchymal lineage. A multi-step culture method was used (Figure 1). First, the hiPSCs were spontaneously differentiated via EB formation for 10 days (D10; Figure 2A). Second, cells outgrew from the EBs for another 10 days (D10+10). During th...

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

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

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

The overall goal of this procedure is to generate chondrogenic lineage cells from human peripheral blood-derived induced pluripotent stem cells, or IPS cells. This method can help answer the key questions in the cartilage regeneration field about using patient derived peripheral blood cells as C cells for the generation of the IPS cells for cartilage repair. The main advantages of this technique are that it is easily reproducible, and the chondrocyte differentiation occurs on the serum and xeno-free conditions.Begin by plating human IPS cells in four milliliters human IPSC medium onto a 60 millimeter tissue culture dish containing a monolayer of irradiated mouse embryonic fibroblast feeder cells. Culture the cells at 37 degrees Celsius in five percent carbon dioxide. When the stem cells are 80 to 90 percent confluent, dissociate the cells with dispace solution for subculturing.After two to three passages, use a fire drawn glass needle to dissect the 80 to 90 percent confluent undifferentiated IPSC colony into smaller fragments, and transfer no more than 100 cell culture pieces into a new nonadherent 100 millimeter petri dish containing 10 milliliters of embryoid body formation medium. Mechanical dissociation is better than automatic digestion because it reduces cell damage to now small fragments of cells to be transferred to the new culture container, and announce manual disposal of the feeder cells, which repress human IPS cell differentiation. Return the human IPS cells to the cell culture incubator.After two days, tilt the dish to let the embryoid bodies settle, and carefully replace three milliliters of the embryoid body formation medium with four milliliters of fresh basal culture medium. After 10 days, embryoid bodies will have formed. Coat a new 100 millimeter dish with four milliliters of 0.1 percent gelatin for 30 minutes at 37 degrees Celsius.Transfer the embryoid bodies with the supernatant into a 15 milliliter conical tube, and let the bodies settle for four to five minutes. Aspirate all but the last 0.5 milliliters of medium, and resuspend the embryoid bodies in 10 milliliters of fresh basal culture medium. When the gelatin has solidified, discard the gelatin.Then seed the cells onto the gelatin-coated dish, and place the dish in the cell culture incubator. After 10 days in culture, the embryoid bodies will have differentiated into fibroblastic cell outgrowths. To induce chondrocyte differentiation, wash the culture with DPBS and detach the cells with three milliliters of 0.25 percent trypsin EDTA at 37 degrees Celsius for five minutes.When the cells have lifted from the bottom of the plate, neutralize the reaction with four milliliters of fresh basal culture medium, and gently titrate the culture five to 10 times. Filter the cell suspension through a 70-micron nylon mesh strainer, and collect the cells by centrifugation. Then seed the cells onto a new gelatin-coated, 100 millimeter plate in 10 milliliters of fresh basal culture medium.When the culture reaches 90 to 100 percent confluency, harvest the cells with three milliliters of 0.25 percent trypsin EDTA as just demonstrated for counting. Transfer a three times 10 to the fifth aliquot of cells into a 15 milliliter polypropylene tube for centrifugation. And resuspend the pellet in one milliliter of chondrogenic differentiation medium.Then, centrifuge the cells again, and place the tube in the cell culture incubator for 21 days without disturbing the pellet. Chondrogenic cells may assemble in vitro, producing characteristic extracellular metrics when under high density culture conditions. After 21 days in culture, a human IPS cell chondrogenic pellet will have formed.After 21 days of culture, the human IPSC chondrogenic cells are positive for alcian blue and toluidine in blue staining, indicating a successful chondrogenic differentiation of the HIPSC pellet. Immunohistochemical analysis for collagen two and collagen 10 further confirms their chondrocyte-like phenotype. Sulfated glycosaminoglycan analysis reveals that the sulfated glycosaminoglycan contents of the cells are significantly upregulated in human IPSC chondrogenic pellets, compared to human IPSC fibroblastic-like cells, embryoid bodies, and undifferentiated human IPS cells.Further, human IPSC chondrogenic cells demonstrate a high gene expression level of chondroprogenitor lineage and fully differentiated chondrocyte markers, suggesting the achievement of a successful chondrogenic differentiation. A multistep culture method used to differentiate human IPSCs into chondrocytes, including spontaneous differentiation via EB formation, cell outgrowth from EBs, monolayer cell culture after cell culture, and 3D pellet culture. The chondrocyte phenotype is assessed by histological analysis, biochemical analysis, and chondrogenic gene expression.After watching this video, you should have a good understanding of how to use a multistep culture method for inducing human IPS cell differentiation into chondrocytes. This technique could be more patient specific and cost effective.

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9.0K visualizações.  Capital Medical University. The overall goal of this procedure is to generate chondrogenic lineage cells from human peripheral blood-derived induced pluripotent stem cells, or IPS cells. This method can help answer the key questions in the cartilage regeneration field about using patient derived peripheral blood cells as C cells for the generation of the IPS cells for cartilage repair. The main advantages of this technique are that it is easily reproducible, and the chondrocyte differentiation occurs on the serum and xe...