Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

Summary

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.

Abstract

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.

Introduction

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 main....

Protocol

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

1. Make 50 mL of hiPSC medium: Knockout Dulbecco's Modified Eagle Medium (DMEM) supplemented with 15% knockout serum replacement (KSR), 5% fetal bovine serum (FBS), 1× nonessential amino acids, 55 μM 2-mercaptoethanol, 2 mM L-glutamine, and 8 ng/mL basic fibroblast growth factor (bFGF).
2. Make 50 mLof EB formation medium: DMEM supplemented with 15% KSR, 5% FBS, 1x nonessential amino acids, 55....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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	TMS-002-C	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
2-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