Chondrogenic differentiation of human induced pluripotent stem cells in a photoclickable biomimetic PEG hydrogel

Abstract

Event Abstract Back to Event Chondrogenic differentiation of human induced pluripotent stem cells in a photoclickable biomimetic PEG hydrogel Elizabeth Aisenbrey1*, Karin Payne2, Ganna Bilousova2 and Stephanie J. Bryant1* 1 University of Colorado, Chemical and Biological Engineering, United States 2 University of Colorado, Anschutz Medical Campus, United States Introduction: Cartilage lacks the ability to regenerate on its own, resulting in the necessity for alternative treatments such as tissue engineering[1]. The overall goal of this study was to develop a tissue engineering strategy for patient-specific repair of cartilage. Induced pluripotent stem cells (iPSCs) are an attractive cell source for cartilage tissue engineering because they can be obtained from a patient in a less invasive way than autologous chondrocytes or bone marrow derived MSCs, and unlike hMSCs, iPSCs have unlimited proliferation potential and the ability to differentiate into any cell in the body[2],[3]. In this study, human iPSCs were encapsulated into a photoclickable cartilage-like biomimetic poly(ethylene glycol) (PEG) hydrogel functionalized with chondroitin sulfate (ChS) and the cell adhesion peptide, RGD. Chondrogenesis was assessed in the cell-laden hydrogels and as a function of two growth factors TGFβ3 alone, BMP2 alone, or together. Materials and Methods: Human skin fibroblasts from a 50 year old female (ATCC) were reprogrammed under low oxygen (5%) conditions via mRNA transfection to generate iPSCs. These iPSCs were induced to become mesenchymal progenitors (iPSC-MPs) and were encapsulated in a hydrogel (9wt% 8-arm PEG(10kDa)norbornene, 1wt% thiolated ChS (16% conjugated), 1.4wt% PEG(1kDa)dithiol, and 0.1mM CYRGDS) via photopolymerization (7 minutes, 352 nm, 5 mW/cm2). The cell-laden hydrogels were cultured for 21 days in chondrogenic differentiation media without and with growth factors (+TGFB3 (2.5ng/ml), +BMP2 (25ng/ml), +TGFB3 (2.5 ng/ml) +BMP2 (25 ng/ml)). The iPSC-MPs were cultured as pellets in identical conditions or with media containing higher concentrations of growth factors ((+TGFB3 (10ng/ml), +BMP2 (100ng/ml), +TGFB3 (10ng/ml) +BMP2 (100ng/ml)). Differentiation was determined by qRT-PCR and immunohistochemistry for chondrogenic specific markers, sox-9, aggrecan, and collagen II and the hypertrophic marker collagen X. Results: After 21 days of culture, the expression of the chondrogenic markers sox9, aggrecan and collagen II were upregulated in the presence of TGFB3 and/or BMP2 compared to the constructs cultured without growth factors. Protein deposition of collagen II and aggrecan were found in all constructs cultured with TGFB3 and/or BMP2, although gene expression shows higher collagen II in constructs only cultured with BMP2. The relative gene expression of the hypertrophic marker collagen X was lower in the hydrogels cultured in BMP2 compared to those cultured in the presence of TGFB3, however, protein deposition was present. Similar results were obtained with the pellet culture when higher concentrations of growth factors were used. Chondrogenesis was not observed at the growth factor concentrations used with the hydrogel. Discussion and Conclusion: This study investigated if iPSCs, a novel cell source for articular cartilage tissue engineering, undergo chondrogenesis when cultured in a cartilage biomimetic hydrogel. The results from this study suggest that the presence of TGFB3 and/or BMP2 enhances chondrogenesis of iPSCs. Interestingly, lower concentrations of growth factors were needed for iPSCs to undergo chondrogenesis when in the hydrogel constructs compared to the pellet culture, suggesting that the biomimetic hydrogel is a promising scaffold for cartilage engineering. Studies are underway to investigate the effect of dynamic compression at physiological conditions on iPSCs. NSF Graduate Research Fellowship; NSF Career Award Grant # 0847390; Academic Enrichment Fund of the University of Colorado School of Medicine