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

Summary

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

Abstract

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.

Introduction

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 cartilage^1,2.

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 MSCs^8,9,10,11.

Various types of growth factors are used to induce chondrogenesis. Usually, BMP and TGFβ family proteins are used, alone or in combination. Differentiation has also been induced with other factors, such as GDF5, FGF2, and IGF1^12,13,14,15. TGFβ1 has been shown to stimulate chondrogenesis in a dose-dependent manner in MSCs¹⁶. Compared to the other isotype, TGFβ3, TGFβ1 induces chondrogenesis by increasing the pre-cartilage mesenchymal cell condensation.TGFβ3 induces chondrogenesis by significantly increasing the mesenchymal cell proliferation¹⁷. However, TGFβ3 is used more frequently by researchers than TGFβ1^7,10,18,19. 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 TGFβ proteins²¹. It has also been shown that BMP2 synergistically enhances the effect of TGFβ3 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 TGFβ3 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.

Protocol

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's Hospital.

1. Chondrogenic Differentiation from iPSCs

1. CBMC-iPSC generation
   1. Generate CBMC-hiPSCs using the protocol shown in our previous work²³.
   2. Collect the blood cells in a 15 mL conical tube and count them using a hemocytometer.
   3. Prepare 3 x 10⁵ cells and centrifuge them for 5 min at 515 x g and RT. Discard the supernatant by suction and resuspend the cells in 0.5 mL of blood cell medium.
   4. Transfer the cells to a well of a non-coated 24-well plate and add the Sendai virus mixture, following the manufacturer's recommendations.
   5. Centrifuge the plate for 30 min at 1,150 x g and 30 °C.
   6. After centrifugation, incubate the cells overnight (O/N) at 37 °C in 5% CO₂.
   7. The next day, transfer the transduced cells to a matrix-coated well. Centrifuge the plate at 1,150 x g for 30 min at 30 °C.
   8. After centrifugation, remove the supernatant, add 1 mL of iPSC medium, and maintain the cells O/N at 37 °C in 5% CO₂.
   9. Maintain the attached cells at 37 °C and 5% CO₂. Change the medium daily, replacing it with fresh iPSC medium.
      NOTE: Colonies will appear on day 14-21 after transduction.
2. EB generation
   1. Maintain the hiPSCs at 37 °C and 5% CO₂. Change the medium daily, replacing it with fresh Essential 8 (E8) medium.
   2. Prepare 2 x 10⁶ hiPSCs in a vitronectin-coated, 100-mm dish in E8 medium.
   3. Remove the E8 medium from the culture dish and wash with sterile phosphate-buffered saline (PBS).
   4. Add 1 mL of 1 mM ethylenediaminetetraacetic acid (EDTA) and incubate at 37 °C and 5% CO₂ for 2 min.
   5. Harvest the cells with 3 mL of E8 medium and transfer them to a new 15-mL conical tube. Centrifuge the cells at 250 x g at room temperature (RT) for 2 min.
   6. Aspirate the supernatant without disturbing the cell pellet and resuspend the cells in 5 mL of E8 medium.
   7. Count the cells using a hemocytometer and prepare 2 x 10⁶ cells for each 100 mm Petri dish. Resuspend the prepared cells in a 10-mL mixture of E8 and EB medium (1:1) with 10 µM rho-associated kinase (ROCK) inhibitor.
   8. Incubate the cells O/N for aggregation at 37 °C and 5% CO₂.
   9. On the following day, harvest the aggregated EBs by pipetting. Centrifuge the cells at 250 x g for 1 min. Remove the supernatant and resuspend the EBs in 10 mL of fresh E8 medium.
   10. Enlarge the generated EBs for 5 days, performing daily changes with fresh E8 medium. For further maturation, change the culture medium to E7 medium. Maintain the EBs at 37 °C and 5% CO₂ for another 5 days, performing daily medium changes with fresh E7 medium.
       NOTE: E7 medium is E8 medium without FGF2.
3. Outgrowth cell induction from EBs
   1. Add 6 mL of 1% gelatin to a 100-mm dish and incubate at 37 °C and 5% CO₂ for 30 min.
   2. Remove the gelatin and dry the dish completely for 2-3 h before use.
   3. Transfer the EBs to a 50-mL conical tube. Allow the EBs to settle to the bottom of the conical tube. Remove the supernatant without disturbing the EBs.
   4. Resuspend the EBs in 10 mL of Dulbecco's Modified Eagle's Medium (DMEM) with 20% fetal bovine serum (FBS). Transfer the EBs to the gelatin-coated, 100-mm dish. Add 10 µM ROCK inhibitor.
   5. Incubate and maintain the cells at 37 °C and 5% CO₂ for 7 days. Change the medium every other day without adding ROCK inhibitor.
4. Chondrogenic pellet formation
   1. Aspirate the culture medium from the dish and wash the cells three times with PBS.
   2. Apply 1 mL of 1 mM EDTA and incubate at 37 °C and 5% CO₂ for 2 min.
   3. Harvest the cells using 5 mL of DMEM with 20% FBS and transfer them to a new 15-mL conical tube.
   4. Centrifuge the cells for 2 min at 250 x g and RT. Discard the supernatant and resuspend the pellet in 10 mL of DMEM with 20% FBS.
   5. Filter and discard the cell clumps using a 40 µm cell strainer and harvest the single cells. Count the single cells using a hemocytometer.
   6. Centrifuge the cells for 2 min at 250 x g and RT. Remove the supernatant and seed 3 x 10⁵ cells per pellet in a 15 mL conical tube with 300 µL of chondrogenic differentiation medium (CDM).
   7. For pellet formation, centrifuge the cells for 5 min at 680 x g and RT.
      NOTE: Pellets are maintained in 15 mL conical tubes during the differentiation of the chondrogenic pellets.
   8. Incubate the cells overnight at 37 °C and 5% CO₂.
   9. Change the CDM every 2-3 days. Within 3 days, pellets will exhibit flattened, spheroidal morphologies. Maintain the pellets for 21 days at 37°C and 5% CO₂.

2. Chondrogenic Pellet Characterization by Staining

1. Chondrogenic embedding
   1. Before embedding, prepare and melt paraffin at 58 °C.
   2. Fix the pellets in 1 mL of 4% paraformaldehyde for 2 h at RT in a 1.5 mL tube.
      Caution: Paraformaldehyde is highly toxic. Avoid contact with eyes, skin, or mucous membranes. Minimize exposure and avoid inhalation while preparing it.
   3. Place one layer of gauze onto the cassette and transfer the fixed pellets using a pipette. Cover the pellet by folding the gauze and close the cassette lid.
   4. Initiate dehydration in 100 mL of 70% ethanol (EtOH) twice, 10 min each. Dehydrate the pellets through sequential 10-min washes in 80% and 95% EtOH.
   5. Transfer the pellets to 100% EtOH for 10 min. Repeat three times with fresh EtOH.
   6. For "clearing," exchange the solution for a 100 mL, 1:1 mixture of EtOH and xylene, followed by a 1:2 mixture of EtOH and xylene for 10 min each. Clear the remaining EtOH by incubating the pellets twice in 100% xylene, 10 min each.
   7. For paraffin infiltration, incubate the pellets in sequential xylene and paraffin mixtures. Perform the whole paraffin infiltration process at 58 °C. Incubate the pellets in 100 mL of a 2:1 mixture of xylene and paraffin for 30 min.
   8. Exchange the solution for 100 mL of a 1:1 mixture of xylene and paraffin and incubate for 30 min.
   9. Exchange the solution for 100 mL of a 1:2 mixture of xylene and paraffin and incubate for 30 min.
   10. For the final infiltration, transfer the pellets to the first bath of 100% paraffin and incubate for 2 h.
   11. Transfer the pellets to the second bath of 100% paraffin and incubate O/N at 58 °C.
   12. The next day, gently transfer the pellets to a mold using a tweezer. Add paraffin to the mold from the paraffin dispenser. Solidify the paraffin for 30 min at 4 °C.
   13. Slice the sections at 7 µm and transfer the sections onto the slide. Allow the slides to dry overnight and store the slides at RT until they are ready for use.
2. Slide preparation
   1. Deparaffinize the slides by moving them through 100 mL of 100%, 90%, 80%, and 70% EtOH sequentially for 5 min each.
   2. Place the slides in a glass jar and rinse with tap water for 5 min.
3. Alcian blue staining
   1. Incubate the slides in 50 mL of 1% alcian blue solution for 30 min.
      NOTE: Alcian blue is diluted in 3% acetic acid solution. Adjust the pH to 2.5 using acetic acid.
   2. Place the slides in a glass jar and rinse with tap water for 2 min.
   3. Rinse the slides in deionized water (DW) and counterstain with nuclear fast red solution for 2 min.
   4. Place the slides in a glass jar and rinse with tap water for 1 min.
   5. 2.3.5)Proceed to dehydrate and mount the slides in step 2.6.
4. Toluidine blue staining
   1. Incubate slides in 50 mL of toluidine blue solution for 4 min.
   2. Place the slides in a glass jar and rinse with tap water for 5 min.
   3. Proceed to dehydrate and mount the slides in step 2.6.
5. Immunohistochemical staining
   1. Incubate the slides in 3% H₂O₂for 15 min for endogenous peroxidase blocking.
   2. Apply 200 µL of primary antibody (anti-COL1A1 and -COL2A1) diluted 1:100 in tris-buffered saline (TBS) containing 1% bovine serum albumin (BSA) and 5% normal goat serum (NGS) to the slides and incubate O/N at 4 °C.
   3. The next day, place the slides in a glass jar and wash them with 50 mL of TBS containing 0.1% polysorbate 20 (TBST) three times for 5 min each.
   4. Apply 200 µL of secondary antibody (goat anti-rabbit IgG antibody, diluted 1:200 in TBS containing 1% BSA and 5% NGS) to the slides and incubate at RT for 40 min.
   5. Place the slides in a glass jar and wash them three times with 50 mL, 5 min each.
   6. For signal amplification, apply HRP-conjugate streptavidin solution to the slides and incubate for 10 min.
   7. Place the slides in a glass jar and wash them three times with 50 mL, 5 min each.
   8. Mix the DAB-peroxidase substrate solution, apply 200 µL of solution to each slide, and incubate for 1 min.
   9. Place the slides in a glass jar and rinse with tap water for 5 min.
   10. Counterstain with Mayer's hematoxylin for 1 min.
   11. Wash the slides with DW.
   12. Proceed to dehydrate and mount the slides in step 2.6.
6. Dehydration and mounting
   1. Dehydrate the slides by moving them sequentially through 100 mL of 70%, 80%, 90%, and 100% EtOH, 30 s each.
   2. Dip the slides into two changes of xylene for 1 min each.
   3. Add 50 µL of mounting solution to the coverslips and place the slides on top.

Results

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 (

Discussion

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 chondrocytes^25,26,27,28

Materials

Name	Company	Catalog Number	Comments
Plasticware
100 mm Dish	TPP	93100
6-well Plate	TPP	92006
50 mL Cornical Tube	SPL	50050
15 mL Cornical Tube	SPL	50015
10 mL Disposable Pipette	Falcon	7551
5 mL Disposable Pipette	Falcon	7543
12-well Plate	TPP	92012
Name	Company	Catalog Number	Description
E8 Medium Materials
DMEM/F12, HEPES	Life Technologies	11330-057	E8 Medium (500 mL)
Sodium Bicarbonate	Life Technologies	25080-094	E8 Medium (Conc.: 543 μg/mL)
Sodium Selenite	Sigma Aldrich	S5261	E8 Medium  (Conc.: 14 ng/mL)
Human Transfferin	Sigma Aldrich	T3705	E8 Medium (Conc.: 10.7 μg/mL)
Basic FGF2	Peprotech	100-18B	E8 Medium  (Conc.: 100 ng/mL)
Human Insulin	Life Technologies	12585-014	E8 Medium (Conc.: 20 μg/mL)
Human TGFβ1	Peprotech	100-21	E8 Medium (Conc.: 2 ng/mL)
Ascorbic Acid	Sigma Aldrich	A8960	E8 Medium  (Conc.: 64 μg/mL)
DPBS	Life Technologies	14190-144
Vitronectin	Life Technologies	A14700
ROCK Inhibitor	Sigma Aldrich	Y0503
Name	Company	Catalog Number	Description
Quality Control Materials
18 mm Cover Glass	Superior	HSU-0111580
4% Paraformaldyhyde	Tech & Innovation	BPP-9004
Triton X-100	BIOSESANG	9002-93-1
Bovine Serum Albumin	Vector Lab	SP-5050
Anti-SSEA4 Antibody	Millipore	MAB4304
Anti-Oct4 Antibody	Santa Cruz	SC9081
Anti-TRA-1-60 Antibody	Millipore	MAB4360
Anti-Sox2 Antibody	Biolegend	630801
Anti-TRA-1-81 Antibody	Millipore	MAB4381
Anti-Klf4 Antibody	Abcam	ab151733
Alexa Fluor 488 goat anti-mouse IgG (H+L) antibody	Molecular Probe	A11029
Alexa Fluor 594 goat anti-rabbit IgG (H+L) antibody	Molecular Probe	A11037
DAPI	Molecular Probe	D1306
Prolong gold antifade reagent	Invitrogen	P36934
4% Paraformaldyhyde	Tech & Innovation	BPP-9004
Tween 20	BIOSESANG	T1027
Bovine Serum Albumin	Vector Lab	SP-5050
Anti-Collagen II antibody	abcam	ab34712	1:100
Alcian blue	Sigma Aldrich	A3157-10G
Fast Green FCF	Sigma Aldrich	F7252-25G
Safranin O	Sigma Aldrich	090m0039v
Nuclear fast red	Americanmastertech	STNFR100
xylene	Duksan	115
Ethanol	Duksan	64-17-5
Mayer's hematoxylin solution	wako pure chemical industries	LAK7534
DAP	VECTOR LABORATORIES	SK-4100
Slide Glass, Coated	Hyun Il Lab-Mate	HMA-S9914
Trizol	Invitrogen	15596-018
Chloroform	Sigma Aldrich	366919
Isoprypylalcohol	Millipore	109634
Ethanol	Duksan	64-17-5
RevertAid First Strand cDNA Synthesis kit	Thermo Scientfic	K1622
Name	Company	Catalog Number	Description
Chondrogenic Differentiation Materials
DMEM	Life Technologies	11885	Chondrogenic media component (500 mL)
Penicilin Streptomycin	Life Technologies	P4333	Chondrogenic media component (Conc.: 1%)
Ascorbic Acid	Sigma Aldrich	A8960	Chondrogenic media component (Conc.: 64 μg/mL)
MEM Non-Essential Amino Acids Solution (100x)	Life Technologies	11140-050	Chondrogenic media component (Conc.: 100 mM)
rhBMP-2	R&D	355-BM-050	Chondrogenic media component (Conc.:100 ng/ml)
Recombinant Hman TGF-beta3	R&D	243-B3-002	Chondrogenic media component (Conc.:10 ng/ml)
KnockOut Serum Replacement	Life Technologies	10828-028	Chondrogenic media component (Conc.: 1%)
ITS+ Premix	BD	354352	Chondrogenic media component (Conc.: 1%)
Dexamethasone-Water Soluble	Sigma Aldrich	D2915-100MG	Chondrogenic media component (Conc.:10-7 M)
GlutaMAX Supplement	Life Technologies	35050-061	Chondrogenic media component (Conc.: 1%)
Sodium pyruvate solution	Sigma Aldrich	S8636	Chondrogenic media component (Conc.: 1%)
L-Proline	Sigma Aldrich	P5607-25G	Chondrogenic media component (40 μg/ml)