The generation and functional characterization of induced pluripotent stem cells from human intervertebral disc nucleus pulposus cells

Abstract

Disc degenerative disease (DDD) is believed to originate in the nucleus pulposus (NP) region therefore, it is important to obtain a greater number of active NP cells for the study and therapy of DDD. Human induced pluripotent stem cells (iPSCs) are a powerful tool for modeling the development of DDD in humans, and have the potential to be applied in regenerative medicine. NP cells were isolated from DDD patients following our improved method, and then the primary NP cells were reprogramed into iPSCs with Sendai virus vectors encoding 4 factors. Successful reprogramming of iPSCs was verified by the expression of surface markers and presence of teratoma. Differentiation of iPSCs into NP-like cells was performed in a culture plate or in hydrogel, whereby skin fibroblast derived-iPSCs were used as a control. Results demonstrated that iPSCs derived from NP cells displayed a normal karyotype, expressed pluripotency markers, and formed teratoma in nude mice. NP induction of iPSCs resulted in the expression of NP cell specific matrix proteins and related genes. Non-induced NP derived-iPSCs also showed some NP-like phenotype. Furthermore, NP-derived iPSCs differentiate much better in hydrogel than that in a culture plate. This is a novel method for the generation of iPSCs from NP cells of DDD patients, and we have successfully differentiated these iPSCs into NP-like cells in hydrogel. This method provides a novel treatment of DDD by using patient-specific NP cells in a relatively simple and straightforward manner.

Keywords: differentiation; disc degenerative disease; induced pluripotent stem cell; nucleus pulposus; reprogram.

Figure 1. Overview of the NP-iPSC generation protocol and immunostaining of specific markers

(A) NPCs transduced with SeV expressing human OCT3/4, SOX2, KLF4 and c-MYC. NPC derived iPS colonies emerge at 14–28 days after NP cell transduction. (B) Staining of Alkaline phosphatase (AP) and pluripotency markers (NANOG, OCT3/4, SOX2, TRA-1–60, TRA-1–81 and SSEA-4) in induced cell colonies

Figure 2. Characterization of iPS colonies generated from NPCs

(A) Real time polymerase chain reaction (RT-PCR) analysis of endogenous and total genes in induced colonies. 1# and 2# represent NPC-derived iPSCs P4 and P16, 3# represent H9 P38. (B) DNA methylation analysis in the promoters of Oct4 and Nanog by bisulfite sequencing. Human NP and H9 cells were used as controls. (C) Karyotyping analysis of a representative colony. (D) Hematoxylin and eosin (HE) staining of teratomas derived from NP-derived iPSCs.

Figure 3. Differentiation analysis of iPSCs

Brightfield image showing the morphology of iPSCs 3 weeks post differentiation (magnification 100×, bar = 100 μm). Toluidine blue staining of glycosaminoglycans in representative iPS cell clusters (magnification 200×, bar = 50 μm). Immunostaining of iPS cell cultures for the presence of collagen II, aggrecan, and CD24. NP cells were used as positive control (magnification 100×, bar = 100 μm). Regions of positive staining are shown as brown (magnification 200×, bar = 50 μm). F-iPS: fibroblast derived iPS cells; NP-iPS: NP cells derived iPS cells; NPC: NP cells; Induction: iPSCs induced with NP differentiation media; Non-induction: iPSCs cultured without NP differentiation media.

Figure 4. Quantitative analysis of NP specific proteins and genes

Protein expressions of collagen II, aggrecan and CD24were analyzed by western blotting. Quantitative analysis was did for collagen II, aggrecan, CD24, KRT18, Basp1 and CD54 gene expression in micromass-cultured iPSCs after 3 weeks differentiation, * compare with undifferentiation group p < 0.01, # compare with NP cells p < 0.05. F-iPS-C: fibroblast derived iPS cells without differentiation; F-iPS-D: fibroblast derived iPS cells cultured with NP differentiation media; NP-iPS-C: NP cells derived iPS cells without differentiation; NP-iPS-D: NP cells derived iPS cells cultured with NP differentiation media.

Figure 5. Differentiation analysis of iPSCs in hydrogel

Toluidine blue staining of glycosaminoglycans in hydrogel and culture plate (magnification 100×, bar = 100 μm). Quantitative analysis was performed for collagen II, aggrecan, CD24, KRT18, Basp1 and CD54 gene expression in in hydrogel and culture plate, * compared with cells in culture plate. 2D: iPSCs differentiated in culture plate, 3D: iPSCs differentiated in hydrogel.

Figure 6. Immunofluorescence analysis of iPSC differentiation in hydrogel

Immunofluorescence staining of iPSCs for the presence of collagen II and CD24. Positive staining is shown by red fluorescence, blue fluorescence is for the nucleus (magnification 200×, bar = 50 μm). 2D: iPSCs differentiated in culture plate, 3D: iPSCs differentiated in hydrogel.