Differentiation of mouse induced pluripotent stem cells (iPSCs) into nucleus pulposus-like cells in vitro

Abstract

A large percentage of the population may be expected to experience painful symptoms or disability associated with intervertebral disc (IVD) degeneration - a condition characterized by diminished integrity of tissue components. Great interest exists in the use of autologous or allogeneic cells delivered to the degenerated IVD to promote matrix regeneration. Induced pluripotent stem cells (iPSCs), derived from a patient's own somatic cells, have demonstrated their capacity to differentiate into various cell types although their potential to differentiate into an IVD cell has not yet been demonstrated. The overall objective of this study was to assess the possibility of generating iPSC-derived nucleus pulposus (NP) cells in a mouse model, a cell population that is entirely derived from notochord. This study employed magnetic activated cell sorting (MACS) to isolate a CD24(+) iPSC subpopulation. Notochordal cell-related gene expression was analyzed in this CD24(+) cell fraction via real time RT-PCR. CD24(+) iPSCs were then cultured in a laminin-rich culture system for up to 28 days, and the mouse NP phenotype was assessed by immunostaining. This study also focused on producing a more conducive environment for NP differentiation of mouse iPSCs with addition of low oxygen tension and notochordal cell conditioned medium (NCCM) to the culture platform. iPSCs were evaluated for an ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Results demonstrated that a CD24(+) fraction of mouse iPSCs could be retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of a hypoxic environment and NCCM induced a similar phenotypic result. In conclusion, this study suggests that mouse iPSCs have the potential to differentiate into NP-like cells and suggests the possibility that they may be used as a novel cell source for cellular therapy in the IVD.

Figure 1. iPSC morphology and the expression notochordal-related transcriptional factors.

(A) Undifferentiated iPSCs cultured on a feeder layer of mitotically inactivated mouse embryonic fibroblasts and formed colonies (top panels); iPSCs formed cell clusters of embryoid bodies (EBs, bottom panels) when promoted to undergo spontaneous differentiation in ultra-low attaching dishes (bars = 100 µm). (B) Relative mRNA levels for transcriptional factors (Oct4, Noggin, Brachyury, Noto, Foxa2, Shh) in EBs (3 days) normalized to undifferentiated iPSCs for each target gene. Data are averages of two replicates for cells collected from multiple EBs and undifferentiated culture.

Figure 2. Enrichment of CD24⁺ cell population from undifferentiated iPSCs via magnetic activated cell sorting (MACS).

(A) General experimental overview. (B) Flow cytometry analysis for the expression of CD24 in presorted, “flow-through” and sorted cell fractions. Undifferentiated iPSCs incubated with CD24 antibody and magnetic beads were passed through a MACS column, yielding a CD24⁺ fraction of cells; in the “flow-through” fraction, ~ 5.1% of total cells on average were found to express CD24, which was less than that in the presorted fraction. The gated readouts represent the percentage of cells expressed CD24 in presorted cells and sorted fractions from one sorting experiment.

Figure 3. Relative mRNA levels in undifferentiated, presorted iPSC populations, “flow-through” and sorted CD24⁺ fractions for notochordal markers (Noto, Shh, Foxa2, Brachyury and Noggin).

Data are expressed as fold-difference (2^-ΔΔCt, n=3 independent sorting experiments, mean+SE) between sorted CD24⁺ or “flow-through” and presorted samples. The relative mRNA level in presorted cells was set as the calibrator (value=1) for normalizing the relative mRNA levels in the CD24⁺ cell fraction and “flow-through” fraction (* p<0.01, ANOVA: significant fold-difference detected between presorted cells and CD24⁺ cells or CD24^- cells in “flow-through”, and between CD24^- cells in “flow-though” and CD24⁺ cells). Differences were detected in all targets except for Noggin between unsorted cells and sorted CD24⁺ cells.

Figure 4. Differentiation of enriched CD24⁺ iPSCs in a 3D culture system in vitro.

(A) Cell constructs form both CD24^- and CD24⁺ fractions extracted from Transwells at the end of culture (day 28) and representative images of histology and immunostaining revealed cell morphology (H and E) and matrix accumulation (Saf O for glycosaminoglycan and type II collagen, Col II; Scale bar: 50 µm). (B) CD24⁺ cell construct and representative images for expression of NP-associated markers by immunostaining (integrin α6, ITGα6; laminin α5, LM α5; vimentin, VIM and cytokeratin 5/8, CK) in cell constructs at day 28. Scale bar: 20 µm).

Figure 5. Differentiation of iPSCs under hypoxic conditions in vitro.

(A) General experimental overview. (B) Sulfated-glycosaminoglycan content normalized to DNA content (sGAG/DNA) in cell constructs (results shown as mean+SD, n=3 samples). Significant effects of treatment and time were detected using a two-way ANOVA and Tukey’s HSD (p<0.05). Treatment and time points denoted by different letters were found to be significantly different: uppercase letters for treatment; lowercase letters for time. (C) Total collagen content normalized to DNA (collagen/DNA) in cell constructs (results shown as mean+SD or –SD, n=3 samples). No significant effects of treatment on collagen production were detected via two-way ANOVA and Tukey’s HSD (p>0.05). (D) Cell constructs extracted from Transwelsl at the end of the culture (day 28) and representative images of histology and immunostaining revealed matrix accumulation (Saf O for glycosaminoglycan and type II collagen, Col II; Scale bar: 100 µm) and expression of NP-associated markers (vimentin, VIM; cytokeratin 5/8, CK and integrin α6, ITGα6) in cell constructs at days 28. Scale bar: 50 µm. NCCM: notochordal cell conditioned medium. (E) Representative images of histology (H and E) and immunostaining for above mentioned NP-associated markers and matrix proteins in the immature mouse IVD (1 month-old) tissue control (AF: anulus fibrosus; NP: nucleus pulposus).