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 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.
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.
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
2. Cell Pellet Formation and Chondrocyte Differentiation
3. Analysis of Chondrogenic Differentiation
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 these two steps, the iPSCs gradually lost their original morphologies and obtained spindle-shaped morphologies (Figure 2B), which then changed to a fibroblastic shape after passage. Third, cells were expanded in monolayer after subculture (Figure 2C). Residual undifferentiated cells were excluded during this step. Then, the cells were expanded and committed to fibroblastic-like cells after 5-7 days in monolayer culture (D10+10+7). Fourth, when the hiPSC-fibroblastic-like cells (hiPSC-F) reached about 90% confluence, they were induced to differentiate into chondrocytes via a 3D pellet culture (Figure 2D)10.
Characterization of hiPSC-Chon Pellets:
hiPSC-F were cultured in 15 mL polypropylene tubes in pellet for 21 days. Chondrogenic cells may assemble in vitro and produce a characteristic extracellular matrix when in high-density culture. At the end of culture, we could see a dense, cartilage-like aggregate, the hiPSC-Chon pellet, which was up to 2-3 mm long and 3 mm thick (Figure 2D). The cells were positive for alcian blue (Figure 3A) and toluidine blue (Figure 3B) staining, which indicated the successful chondrogenic differentiation of hiPSC pellets. Immunohistochemistry analysis for collagen II (Figure 3C) and collagen X (Figure 3D) further proved that the hiPSC-Chon pellets had developed a chondrocyte-like phenotype. Negative controls of immunohistochemistry for collagen II and collagen X were performed to better prove the positive staining (data not shown)5.
sGAG analysis was also performed after chondrogenic differentiation (Figure 3E). sGAG contents were detected in hiPSC-Chon pellets, hiPSC-F, EBs, and the undifferentiated hiPSCs. sGAG content was significantly upregulated in hiPSC-Chon pellets than in the other groups (P <0.05). In the positive control of hMSC-Chon, the sGAG content was also significantly upregulated (P < 0.05) compared to hMSCs. However, the sGAG contents between hMSC pellets and hiPSC-Chon pellets demonstrated no difference (P >0.05).
Gene Expression of Chondrogenic Differentiation Markers:
Gene expression of the differentiation markers for the chondro-progenitor lineage (SOX9 and COL2) and fully-differentiated chondrocytes (AGGRECAN and COL10) were used to characterize the phenotype of the chondrogenic pellets (Figure 4). In comparisons between hiPSCs, hiPSC-F, and hiPSC-Chon pellets, expressions of COL2, COL10, SOX9, and AGGRECAN were significantly upregulated in hiPSC-Chon than in the other groups (P <0.05). In the positive control of hMSC-Chon, the expression of these markers was also significantly upregulated than in hMSCs (P < 0.05). However, the gene expression between hMSC pellets and hiPSC-Chon pellets demonstrated no differences (P >0.05). In all, these results suggest a successful chondrogenic differentiation process from human iPSCs.
Figure 1: Schematic Overview of the Protocol. A multi-step culture method used to differentiate human iPSCs into chondrocytes, including: 1) spontaneous differentiation via EB formation, 2) cell outgrowth from EBs, 3) monolayer cell culture after subculture, and 4) 3D pellet culture. The chondrocyte phenotype is assessed by histological analysis, biochemical analysis, and chondrogenic gene expression. Please click here to view a larger version of this figure.
Figure 2: Generation of Chondrocytes from hiPSCs. (A) EB formation on D10. Scale bar = 100 µm. (B) Cell outgrowth from EBs on D10+10. Scale bar = 100 µm. (C) Monolayer cell culture on D10+10+7. Scale bar = 100 µm. (D) 3D pellet culture. This figure has been modified from our previous study5. Please click here to view a larger version of this figure.
Figure 3: Characterization of hiPSC-Chon Pellets. (A) Alcian blue staining and (B) toluidine blue staining of glycosaminoglycans and proteoglycans. Scale bar = 100 µm. (C and D) Immunohistochemistry for collagen II and collagen X. Scale bar = 100 µm. (E) Biochemical characterization of hiPSC-Chon pellets versus hiPSCs, EBs, and hiPSC-F compared with hMSC-Chon versus hMSCs. sGAG per DNA. The bar represents the mean ± SEM. N = 3, *P < 0.05. This figure has been modified from our previous study5. Please click here to view a larger version of this figure.
Figure 4: Gene Expression Analysis. RT-qPCR gene expression analysis of chondrogenic differentiation markers (COL2, COL10, SOX9,and AGGRECAN) in hiPSC-Chon versus hiPSCs and hiPSC-F compared with hMSC-Chon versus hMSCs. The bar represents the mean ± SEM. N = 3, *P <0.05. This figure has been modified from our previous study5. Please click here to view a larger version of this figure.
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 field12,13. The reprogramming efficiency of generating integration-free iPSCs from blood cells in this study was satisfied. More than 30 iPSCs could be produced from 2 mL of peripheral blood. Therefore, PBCs have the potential to be the seed cells used to generate iPSCs for cartilage engineering and other clinical applications.
The main steps of chondrogenic differentiation from hiPSCs included: EB formation, cell outgrowth from EBs, monolayer culture, and 3D pellet culture. Undifferentiated hiPSC colonies are dissected into smaller pieces using a fire-drawn glass needle. The mechanical method, although more technical, is better than enzymatic digestion (such as dispase or collagenase) because of the reduced damage and the specific size (50 – 100 µm in diameter) at acquisition. Furthermore, mechanical digestion can manually dispose of the feeder cells, which will repress the hiPSC differentiation. hiPSCs spontaneously differentiate to form EBs, which are characterized as the three-dimensional, multi-cellular aggregates with smooth borders. Several EBs can cluster together to form irregular shapes. In order to maintain the EBs in good conditions, less than 100 EBs are cultured in a 100 mm, non-adherent Petri dish, with 10 mL of EB formation medium. About 50 EBs in one 100 mm dish is thought to be the best concentration. EBs are then seeded onto 10 cm, gelatin-coated dishes with basal culture medium. The density of the distribution of EBs is important for the sufficient outgrowth of EBs. Less than 100 EBs are cultured onto a 100-mm dish. Within 10 days of culture, fibroblastic cells are gradually outgrown and expanded from the EBs. The monolayer step is performed to exclude residual undifferentiated cells present in the EBs, as well as to expand cells committed to the mesenchymal lineage. 0.5-1 x 106 cells are seeded in a 100-mm dish for monolayer cell culture. The expression of cell surface markers on hiPSC-F were analyzed by flow-cytometric analysis in our previous study5. The results showed that the majority of hiPSC-F expressed CD73 (81.81 ± 2.05%) and CD105 (endoglin; 81.90 ± 1.61%), which are known to be the positive human mesenchymal markers. Furthermore, six different iPSCs and one human embryonic stem cell (ESC) have been used to reproduce these methods.
The induced chondrogenic differentiation of pluripotent cells is also a complex key process. In view of this, classic chondrogenic medium was utilized for the induction of chondrogenesis from hiPSCs. TGF-beta1 and dexamethasone are supplemented in the pellet culture medium. These factors have been demonstrated to have a significant influence on chondrogenic potential abilities14. Another difference from other protocols was the concentration of 10% ITS, which is much higher than the typically reported 1% ITS15,16. 1% ITS plus 10% FBS enhanced cartilage formation in other methods17,18. ITS as a serum substitute can promote chondrocyte proliferation and formation and retain the chondrogenic phenotypes. In order to replace the animal components of FBS, we upgraded the concentration of ITS to 10%, which has been proved to efficiently promote chondrocyte differentiation7.
A high-density cell culture is another essential factor for chondrogenic differentiation. There are many other cell culture methods that can be used to induce chondrogenic differentiation, such as micromass culture, co-culture with other cells, biomaterial-based culture, and genetic manipulation1,19,15. The 3D pellet culture in our study, which results in a high cell density and high cell-cell interaction, is easier to perform without other cells or materials. Since it is performed in the 15 mL centrifuge tubes, one limitation is that it can only be used in a small-scale chondrogenic differentiation assay. However, 96-well plates with round bottoms could be used as a promising alternative7. Therefore, other improvement to the culture methods could promote the efficiency of chondrogenic differentiation in vitro. In our study, the chondrogenic induction for as long as 21 days was done under serum-free and xeno-free condition, during which all animal-related components were removed. Therefore, the procedure in our study is adaptable for future clinical applications.
It is believed that autologous stem cells would be the ideal choice for cartilage repair, as they may not only decrease rejection, but also achieve tissue regeneration by taking advantage of the natural course of embryonic development20,21. However, they were found to have limited proliferative potential in vitro22. Therefore, an integration-free method for generating chondrocytes from PBCs via iPSCs may be a more promising approach for cartilage tissue engineering. With our method, 2 mL of blood could be enough to induce the patient-specific chondrocytes needed for cartilage defects. Moreover, we also used hMSCs as a positive control to compare with cells differentiated from iPSCs, which suggested that iPSCs have good chondrogenic differentiation potentials.
In conclusion, this study proved that PBCs can be used as candidates for chondrocyte regeneration. This could further reflect a future direction to generate seed cells for cartilage repair in a patient-specific and cost-effective approach to regenerative medicine.
The authors have nothing to disclose.
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.).
Knockout DMEM | Invitrogen | 10829018 | Basal medium used for hiPSC culture and EB formation medium |
Knockout Serum Replacement (KSR) | Invitrogen | 10828028 | A more defined, FBS-free medium supplement used for hiPSC culture and EB formation medium |
Fetal bovine serum (FBS) | Hyclone | sh30070.03 | Used for hiPSC culture and EB formation medium,offers excellent value for cell culture |
Nonessential amino acids | Chemicon | TMS-001-C | Used as a growth supplement in all the cell culture medium, to increase cell growth and viability |
L-glutamine | Invitrogen | 35050061 | An amino acid required for cell culture |
Basic fibroblast growth factor (bFGF) | Peprotech | 100-18B | A cytokine used for sustaining the pluripotency and self-renewal of hiPSCs |
Dispase | Invitrogen | 17105041 | Used for hiPSC dissociation for subculture |
DMEM | Gibco | C11960 | Basal medium used for MSC culture medium |
0.1% gelatin | Millipore | ES-006-B | Used for cell attachment onto the dishes |
0.25% trypsin/EDTA | Gibco | 25200072 | Used for cell dissociation |
DPBS | Gibco | 14190250 | A balanced salt solution used for cell wash or reagent preparing |
β-mercaptoethanol | invitrogen | 21985023 | Used as a growth supplement in all the cell culture medium. |
ITS | invitrogen | 41400045 | Insulin, Transferrin, Selenium Solution.Used for chondrogenic differentiation. |
Ascorbic acid | Sigma | 4403 | Known as vitamin C. It helps in active growth and has antioxidant property. |
Sodium pyruvate | Gibco | 11360070 | Added to cell culture medium as an energy source in addition to glucose. |
Transforming growth factor-beta 1 | Peprotech | AF-100-21C | A cytokine that regulate cell proliferation, growth and chondrogenic differentiation. |
Rabbit polyclonal antibodies against Collagen II | Abcam | ab34712 | This antibody reacts with Type II collagens,which is specific for cartilaginous tissues. |
Mouse monoclonal antibodies to Collagen X | Abcam | ab49945 | This antibody reacts with Type X collagen,which is a product of hyperthrophic chondrotocytes. |
Permount | Fisher Scientific | SP15-100 | For mounting and long-term storage of slides |
Toluidine blue | Sigma | 89640 | Used for proteoglycans detection. |
Alcian blue | Amresco | #0298 | Used for glucosaminoglycans detection. |
Papain | Sigma | P4762-25MG | Used to digest chondrogenic pellets. |
Dimethylmethylene blue | Sigma | 341088-1G | Used to quantitate glycosaminoglyans |
Chondroitin sulfate sodium salt from shark cartilage | Sigma | C4384-250MG | Used to draw the standard curve for sGAG content measurement. |
Qubit dsDNA HS assay kit | Invitrogen | Q32851 (100) | Used to determine DNA content |
TRIzol | Invitrogen | 15596018 | Used for RNA isolation from cells |
Reverse Transcriptase System | Promega | A3500 | Used to convert RNA into cDNA |
SYBR FAST qPCR kit Master Mix | Kapa | KK4601 | Used for Real-time PCR |