Differentiation of Human-Induced Pluripotent Stem Cells (iPSCs) on Laminin-functionalized PEG Hydrogels under Nucleus Pulposus-Like Culture Conditions

Abstract

Introduction Cell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration1-4; however, few viable sources of cells have been identified. Induced pluripotent stem cells (iPSCs) are an attractive cell source since they are derived from the patient's somatic cells,5 thereby providing an autologous source of cells for IVD regeneration. We have previously demonstrated that mouse and human iPSCs can differentiate into nucleus pulposus (NP)-like cells in vitro when cultured in a laminin-rich (Matrigel) system.6 To provide for a more controlled microenvironment, we have developed an injectable, laminin-111 functionalized poly (ethylene glycol) (PEG-LM111) hydrogel for IVD cell-mediated regeneration.6 The stiffness and laminin density of this PEG-LM111 hydrogel was previously optimized to promote or maintain the expression of immature NP-specific markers for primary NP cells in vitro.7 The goal of this study is to evaluate if human iPSCs can be promoted to similarly express markers specific to the immature NP cell when cultured upon this PEG-LM111 hydrogel, toward the goal of promoting generation of native NP-like tissue from stem cells in vitro. Materials and Methods Cell Generation iPSCs were generated from human embryonic dermal fibroblasts through transient inducible overexpression of transcription factors (OCT4, SOX2, KLF4, and MYC) by a lentiviral-based gene delivery system (a polycystronic vector with the reverse tetracycline transactivator (M2rtTA) and doxycycline). Selected iPSC colonies were maintained in culture upon a primary mouse embryo fibroblasts (PMEF) feeder layer. As we reported previously, iPSC colonies expressed a subset of human pluripotent cell markers (i.e., OCT4, SOX2, SSEA-4, TRA1-60, TRA1-81, and alkaline phosphatase), NP-associated integrins (α3, α6, and β1 subunit), NP-specific molecular markers (CD24 and Brachyury), as well as MSC markers (CD29 and CD90).9 Cell Differentiation “Soft” PEG-LM111 (2% PEG-diacrylate prepared with chemically coupled 1 mg/mL laminin-111/PEG; ∼ 200 Pa) hydrogels were UV-crosslinked in Transwell inserts. Undifferentiated iPSCs were seeded (106 cells/insert) on PEG-LM111 substrates in in each Transwell and cultured in notochordal cell conditioned medium (NCCM) from porcine NP tissue (DMEM based, 1% ITS10) under 2% hypoxia condition;11 undifferentiated iPSCs cultured under equivalent conditions but with NP differentiation medium (DMEM/F12, ITS, NEAA9) was used as a control. Immunohistochemistry Differentiated cells were harvested for cryosectioning at 10 or 21 days of culture. Cell morphology and proteoglycan synthesis was assessed by histological staining (H&E and Safranin O). Expression of NP markers was evaluated by immunostaining for matrix proteins (type II collagen [COL II], laminin 10 [LM511]), laminin related receptors (CD239, integrin subunits α6, β4), a subset of NP-specific markers (cytokeratin 8 [KRT8], N-cadherin, CD24), and some non-NP-markers (type I collagen, E-cadherin). Results On the “soft” laminin-presenting substrate, differentiated cells expressed collagen II but not collagen I at both 10 and 21 days, which is characteristic of the NP cell phenotype (Figs. A and B). NCCM promoted expression of NP-specific markers (LM511, CD239, cytokeratin 5/8, Fig. B) in the differentiated cells when cultured on “soft” PEG-LM111 gels, as compared with NP differentiation medium (Fig. A). Differentiated cells also stained positively for CD24 (Fig.), proteoglycans (by Safranin O) and N-cadherin under both culture medium conditions (data not shown). It is noteworthy that other laminin receptors (integrin subunits α6, β4) and E-cadherin were not detected in the differentiated iPSCs under either culture medium conditions (data not shown). Fig. Immunostaining for NP markers(Col II, LM511, CD239, CD24, KRT8) in differentiated iPSCs cultured for 21 days in a PEG-LM111 hydrogel system under (A) NP differentiation medium (NPDM) and (B) notochordal cell conditioned medium (NCCM) from porci e NP tissue (bar = 50 μm). Conclusion Human iPSCs have the potential to differentiate toward an NP-like phenotype when cultured on laminin presenting hydrogels and culture conditions similar to native NP tissue environment. It suggests the possibility that they may be used as a novel cell source for cellular therapy in the IVD. Ongoing studies are focusing on selection of NP progenitor cells with notochordal lineage potential from nonvirally transformed human iPSCs.