Name: chondrogenic pellet formation from cord blood-derived induced pluripotent stem cells
Uploaded: 2017-06-19T15:00:00
Description: Watch this Scientific Journal Video about Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells at JoVE.com

This work was supported by a grant from the Korea Healthcare Technology R&#38;D project, Ministry for Health, Welfare &#38; Family Affairs, Republic of Korea (HI16C2177).

This protocol was approved by the institutional review board of the Catholic University of Korea (KC12TISI0861). CBMCs used for reprogramming were directly obtained from the Cord Blood Bank of the Seoul St. Mary&#39;s Hospital.
1. Chondrogenic Differentiation from iPSCs
<ol>
	<li>CBMC-iPSC generation
	<ol>
		<li>Generate CBMC-hiPSCs using the protocol shown in our previous work23.</li>
		<li>Collect the blood cells in a 15 mL conical tube and count th...

This protocol successfully generated hiPSCs from CBMCs. We reprogrammed CBMCs to hiPSCs using a Sendai viral vector containing Yamanaka factors24. Three cases were used in differentiation, and all experiments successfully generated chondrogenic pellets using this protocol. Numerous studies have reported protocols for the differentiation of hiPSCs into chondrocytes25,26,27,28</...

The use of hiPSCs represents a new strategy for drug screening and mechanistic studies of various diseases. From a regenerative perspective, hiPSCs are also a potential source for the replacement of damaged tissues that have limited healing ability, such as articular cartilage1,2.
The regeneration of native articular cartilage has been a challenge for several decades. Articular cartilage is a soft, white tissue that coats the end of bones, protecting them from friction. However, it has limited regenerative ability when damaged, which makes self-repair almost impossible. Therefore, research focused on cartilage regeneration has been ongoing for several decades.
Previously, in vitro differentiation into the chondrogenic lineage was usually performed with BMSCs or native chondrocytes isolated from the knee joint3. Due to their chondrogenic potential, BMSCs and native chondrocytes have numerous merits supporting their use in chondrogenesis. However, because of their limited expansion and unstable phenotype, these cells face several limitations in the reconstruction of articular cartilage defects. Under in vitro culture conditions, these cells tend to lose their own characteristics after 3-4 passages, which eventually affects their differentiation abilities4. Also, in the case of native chondrocytes, additional damage to the knee joint is inevitable when obtaining these cells.
Unlike BMSCs or native chondrocytes, hiPSCs can indefinitely expand in vitro. With the proper culture conditions, hiPSCs have great potential as a replacement source for chondrogenic differentiation. However, it is challenging to change the intrinsic characteristics of hiPSCs5. Moreover, it takes several complicated in vitro steps to direct the fate of hiPSCs to a specific cell type. Despite these complications, the use of hiPSCs is still recommended due to their high self-renewal abilities and their capacity to differentiate into targeted cells, including chondrocytes6.
Chondrogenic differentiation is usually done with three-dimensional culture systems, such as the pellet culture or micromass culture, using MSC-like progenitor cells. If using hiPSCs, the protocol to generate MSC-like progenitor cells differs from the existing protocols. Some groups use monolayer culture of hiPSCs to directly convert the phenotype into MSC-like cells7. However, most studies use EBs to generate outgrowth cells that resemble MSCs8,9,10,11.
Various types of growth factors are used to induce chondrogenesis. Usually, BMP and TGF&#946; family proteins are used, alone or in combination. Differentiation has also been induced with other factors, such as GDF5, FGF2, and IGF112,13,14,15. TGF&#946;1 has been shown to stimulate chondrogenesis in a dose-dependent manner in MSCs16. Compared to the other isotype, TGF&#946;3, TGF&#946;1 induces chondrogenesis by increasing the pre-cartilage mesenchymal cell condensation.TGF&#946;3 induces chondrogenesis by significantly increasing the mesenchymal cell proliferation17. However, TGF&#946;3 is used more frequently by researchers than TGF&#946;17,10,18,19. BMP2 enhances the expression of genes related to the chondrogenic matrix components in human articular chondrocytes under in vitro conditions20. BMP2 increases the expression of genes critical to cartilage formation in MSCs in combination with TGF&#946; proteins21. It has also been shown that BMP2 synergistically enhances the effect of TGF&#946;3 through the Smad and MAPK pathways22.
In this study, CBMC-hiPSCs were aggregated into EBs using EB medium in a low-attachment Petri dish. Outgrowth cells were induced by attaching the EBs to a gelatin-coated dish. Chondrogenic differentiation using outgrowth cells was performed by pellet culture. Treatment with both BMP2 and TGF&#946;3 successfully condensed the cells and induced extracellular matrix (ECM) protein accumulation for chondrogenic pellet formation. This study suggests a simple yet efficient chondrogenic differentiation protocol using CBMC-hiPSCs.

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			<td style="width:247px;">E8 Medium&#160; (Conc.: 100 ng/mL)</td>
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			<td style="width:247px;">E8 Medium (Conc.: 2 ng/mL)</td>
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			<td height="20" style="height:20px;width:207px;">18 mm Cover Glass</td>
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			<td height="20" style="height:20px;width:207px;">Anti-TRA-1-81 Antibody</td>
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			<td style="width:247px;"></td>
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		<tr height="20">
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			<td style="width:113px;">Vector Lab</td>
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			<td style="width:247px;"></td>
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			<td>abcam&#160;</td>
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			<td style="width:247px;">1:100</td>
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			<td>A3157-10G</td>
			<td></td>
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			<td style="width:113px;">Sigma Aldrich</td>
			<td>F7252-25G</td>
			<td></td>
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			<td style="width:113px;">Sigma Aldrich</td>
			<td style="width:87px;">090m0039v</td>
			<td></td>
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			<td>STNFR100&#160;</td>
			<td></td>
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			<td style="width:87px;">115&#160;</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Ethanol</td>
			<td style="width:113px;">Duksan</td>
			<td style="width:87px;">64-17-5</td>
			<td></td>
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			<td height="34" style="height:34px;width:207px;">Mayer&#39;s hematoxylin solution</td>
			<td style="width:113px;">wako pure chemical industries</td>
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			<td></td>
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			<td>SK-4100</td>
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			<td style="width:87px;">HMA-S9914</td>
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			<td style="width:113px;">Invitrogen</td>
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			<td style="width:247px;"></td>
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			<td style="width:113px;">Sigma Aldrich</td>
			<td style="width:87px;">366919</td>
			<td style="width:247px;"></td>
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			<td height="20" style="height:20px;width:207px;">Isoprypylalcohol</td>
			<td style="width:113px;">Millipore</td>
			<td style="width:87px;">109634</td>
			<td style="width:247px;"></td>
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			<td style="width:247px;"></td>
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		<tr height="39">
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			<td style="width:113px;">Company</td>
			<td style="width:87px;">Catalog Number</td>
			<td style="width:247px;">Description</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:207px;">Chondrogenic Differentiation Materials</td>
			<td style="width:113px;"></td>
			<td style="width:87px;"></td>
			<td style="width:247px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:207px;">DMEM</td>
			<td style="width:113px;">Life Technologies</td>
			<td style="width:87px;">11885</td>
			<td style="width:247px;">Chondrogenic media component (500 mL)</td>
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		<tr height="34">
			<td height="34" style="height:34px;width:207px;">Penicilin Streptomycin</td>
			<td style="width:113px;">Life Technologies</td>
			<td style="width:87px;">P4333</td>
			<td style="width:247px;">Chondrogenic media component (Conc.: 1 %)</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:207px;">Ascorbic Acid</td>
			<td style="width:113px;">Sigma Aldrich</td>
			<td style="width:87px;">A8960</td>
			<td style="width:247px;">Chondrogenic media component (Conc.: 64 &#956;g/mL)&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:207px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td style="width:113px;">Life Technologies</td>
			<td style="width:87px;">11140-050</td>
			<td style="width:247px;">Chondrogenic media component (Conc.: 100 mM)</td>
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		<tr height="20">
			<td height="20" style="height:20px;">rhBMP-2</td>
			<td>R&#38;D</td>
			<td>355-BM-050</td>
			<td>Chondrogenic media component (Conc.:100ng/ml)</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Recombinant Hman TGF-beta3</td>
			<td>R&#38;D</td>
			<td>243-B3-002</td>
			<td>Chondrogenic media component (Conc.:10ng/ml)</td>
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		<tr height="34">
			<td height="34" style="height:34px;">KnockOut Serum Replacement</td>
			<td style="width:113px;">Life Technologies</td>
			<td>10828-028</td>
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		<tr height="34">
			<td height="34" style="height:34px;">ITS+ Premix</td>
			<td>BD</td>
			<td>354352</td>
			<td style="width:247px;">Chondrogenic media component (Conc.: 1 %)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dexamethasone-Water Soluble&#160;</td>
			<td style="width:113px;">Sigma Aldrich</td>
			<td>D2915-100MG</td>
			<td>Chondrogenic media component (Conc.:10-7 M)</td>
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		<tr height="34">
			<td height="34" style="height:34px;">GlutaMAX Supplement</td>
			<td style="width:113px;">Life Technologies</td>
			<td>35050-061</td>
			<td style="width:247px;">Chondrogenic media component (Conc.: 1 %)</td>
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		<tr height="34">
			<td height="34" style="height:34px;">Sodium pyruvate solution</td>
			<td style="width:113px;">Sigma Aldrich</td>
			<td>S8636</td>
			<td style="width:247px;">Chondrogenic media component (Conc.: 1 %)</td>
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		<tr height="20">
			<td height="20" style="height:20px;">L-Proline</td>
			<td style="width:113px;">Sigma Aldrich</td>
			<td>P5607-25G</td>
			<td style="width:247px;">Chondrogenic media component (40&#956;g/ml)</td>
		</tr>
	</tbody>
</table>

chondrogenic pellet formation from cord blood-derived induced pluripotent stem cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

In this study, we generated chondrogenic pellets from CBMC-hiPSCs by inducing outgrowth cells from EBs. Chondrogenic differentiation was induced using CBMC-hiPSCs with confirmed high pluripotency11. A simple scheme of our protocol is shown in Figure 1A. Before differentiation, iPSC colonies were expanded (Figure 1B). The expanded iPSCs were assembled as EBs to initiate differentiation (<strong class="xfig"...

Watch this Scientific Journal Video about Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells at JoVE.com

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

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

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

The overall goal of this procedure is to generate chondrogenic pellets from cord blood-derived induced pluripotent stem cells. With this protocol, we can generate a relatively large quantity of chondrogenic pellets. The generated chondrogenic pellets can be used for studies or disease modeling, drug screening, and researcher to medicine to farther our understanding of the nature of cartilage.The human cartilage last year belated to repair. Therefore, cartilage degeneration must be treated by conservative treatment. With unlimited self renew-ability and multi potency, human iPSCs have been highlighted as new replacement cell source for cartilage repair.Demonstrating the procedure will be Eugene Nam and Arie Alice Ream who are graduate students from my laboratory. To begin the protocol, collect blood cells in a 15 milliliter conical tube, and count them using a hemocytometer. Prepare three times 10 to the fifth cells and centrifuge them for five minutes at 515 times G at room temperature.Discard the supernatant by suction, and re suspend the cells in 0.5 milliliters of blood cell medium. Next, transfer the cells to a well on a non-coated, 24 well plate, and add the Sendai virus mixture following the manufacturer's recommendations. Centrifuge the plate for 30 minutes at 1, 150 times G, and 30 degrees celsius.After centrifugation, incubate the cells overnight. The next day, transfer the transude cells to a matrix coded well. Centrifuge the plate.After centrifugation, remove the supernatant, add one milliliter of iPSC medium, and maintain the cells. Change the medium daily, and replace it with fresh iPSC medium. Maintain the human induce pluripotent stem cells, or HIPSCs.Change the medium daily, replacing it with fresh essential eight, or E8 medium. Prepare two times 10 to the 6th HIPSCs in a vitronectin encoded 100 milliliter and E8 medium. Next, remove the E8 medium from the culture dish and wash the cells with sterile phosphate buffered saline, or PBS.Add one milliliter of one millimolar of EDTA, and incubate the dish for two minutes. Then harvest the cells with three milliliters of E8 medium, and transfer them to a new 15 milliliter conical tube. Centrifuge the cells at 250 times G at room temperature for two minutes.Aspirate the supernatant without disturbing the cell palate, and re suspend the cells in five milliliters of E8 medium. After counting the cells with a hemocytometer, prepare two times 10 the 6th cells for each 100 millimeter Petri dish. Re suspend the prepared cells in a 10 milliliter one to one mixture of E8 and EB medium, with 10 micromolar row associated carnase, or rock inhibitor.Incubate the cells overnight for aggregation. On the following day, harvest the aggregated EBs by pipeting. Remove the supernatant, and re suspend the EBs in 10 milliliters of fresh E8 medium.Enlarge the generated EBs for five days, performing daily changes with fresh E8 medium. For further maturation, change the culture medium to E7 medium. Maintain the EBs at 37 degrees celsius and 5%carbon dioxide for another five days, performing daily medium changes with fresh E7 medium.Add six milliliters of 1%gelatin to a 100 millimeter dish, and incubate the dish for 30 minutes. Remove the gelatin, and dry this dish completely for two to three hours before use. Transfer the EBs to a 50 milliliter conical tube.Allow the EBs to settle to the bottom of the conical tube. Remove the supernatant without disturbing the EBs. Next, re suspend the EBs in 10 milliliters of pre-warmed shilbecco's modified equals medium, or DMEM, with 20%fetal bulbing serum, or FBS.Transfer the EBs to the gelatin coating 100 millimeter dish. Add 10 micromolar of rock inhibitor. Incubate and maintain the cells for seven days.Change the medium every other day without adding rock inhibitor. Aspirate the culture medium from the dish. Immediately apply one milliliter of one millimolar EDTA to the dish, and incubate the cells for two minutes.Next, harvest the cells using five milliliter of DMEM with 20%FBS, and transfer them to a new 15 milliliter conical tube. Centrifuge the cells for two minutes at 250 time G, at room temperature. Discard the supernatant, and re suspend the pellet in 10 milliliter of DMEM with 20%FBS.Filter and discard the cell clumps using a 40 micrometer cell strainer, and harvest the single cells. Count the single cells using a hemocytometer. Centrifuge the cells.Remove the supernatant, and seed three times 10 to the fifth cells per pellet in a 15 milliliter conical tube with 300 microliters of chondrogenic differentiation medium, or CDM. For pellet formation, centrifuge the cells, then incubate the cells overnight. Within three days, pellets will exhibit flattened spheroidal morphologies.Maintain the pellets for 21 days. Before embedding, prepare and melt paraffin at 58 degrees celsius. Fix the pellets in one milliliter of 4%paraformaldehyde for two hours at room temperature in a 1.5 milliliter tube.Place one layer of gauze onto the cassette and transfer the fixed pellets using a pipette. Cover the pellet by folding the gauze and closing the cassette lid. Initiate dehydration in 100 milliliters of 70%ethanol.Dehydrate the pellets through sequential terminate washes in 80%and 95%ethanol. Transfer to pellets to 100%ethanol for 10 minutes. For clearing, exchange the solution with a 100 milliliter one to one mixture of ethanol and xylene for 10 minutes.Next, exchange the one to one solution with a one to two mixture of ethanol and xylene for 10 minutes. Clear the remaining ethanol by incubating the pellets in 100%xylene. For paraffin infiltration, incubate the pellets in sequential xylene and paraffin mixtures.Perform the whole paraffin infiltration process at 58 degrees celsius. Incubate the pellets in 100 milliliters of a two to one mixture of xylene and paraffin for 30 minutes. Next, exchange the solution for 100 milliliters of a one to one mixture of xylene and paraffin and incubate for 30 minutes.Then exchange the solution for 100 milliliters of a one to two mixture of xylene and paraffin and incubate for 30 minutes. For the final infiltration, transfer the pellets to the first bath of 100%paraffin and incubate the pellet for two hours. Transfer the pellets to the second bath of 100%paraffin and incubate overnight at 58 degrees celsius.The next day, use tweezers to gently transfer the pellets to a mold. Add paraffin to the mold from the paraffin dispenser. Solidify the paraffin for 30 minutes at four degrees celsius.Slice the sections at seven micrometers and transfer the sections onto the slide. Allow the slides to dry overnight, and store the slides at room temperature until they are ready for use. Before differentiation, iPSC colonies were expanded.The expanded iPSCs were assembled as EBs to initiate differentiation. The generated EBs were attached to gelatin coated dishes to induce outgrowth cells. After 21 days of differentiation, small bead like chondrogenic pellets were obtained and used for further characterization.The quality of the chondrogenic pellets were histologically evaluated on day 21, with BMSC chondrogenic pellets used as the positive control. The accumulation of ECM proteins secreted by the differentiated chrondrocites was confirmed by Alcian blue, in Toluiodine blue staining. Chondrogenic pellets generated from CDMC HIPSCs expressed higher COL2A1 than CBMSC HIPCs derived pellets.The expression of collagen type one, a marker for fibrotic cartilage, was lower in CBMC HIPSC derived pellets compared to the expression of COL2A1. The gene expression of cartilage extracellular matrix proteins on day 21 chondrogenic pellets was confirmed by real time PCR. The aggracan expression of CBMC HIPSC derived pellets was similar to BMSC derived pellets.We provided a detailed method for the differentiation of cord blood mononuclear cell derived iPSCs into chrondrogenic pellets. Once mastered, this technique can be done in two months if it is performed properly. After watching this video, you should have a good understanding of how to generate chondrogenic pellets from iPSCs.

Research

JoVE Journal

Developmental Biology

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