Early intervention for spinal cord injury with human induced pluripotent stem cells oligodendrocyte progenitors

Abstract

Induced pluripotent stem (iPS) cells are at the forefront of research in regenerative medicine and are envisaged as a source for personalized tissue repair and cell replacement therapy. Here, we demonstrate for the first time that oligodendrocyte progenitors (OPs) can be derived from iPS cells generated using either an episomal, non-integrating plasmid approach or standard integrating retroviruses that survive and differentiate into mature oligodendrocytes after early transplantation into the injured spinal cord. The efficiency of OP differentiation in all 3 lines tested ranged from 40% to 60% of total cells, comparable to those derived from human embryonic stem cells. iPS cell lines derived using episomal vectors or retroviruses generated a similar number of early neural progenitors and glial progenitors while the episomal plasmid-derived iPS line generated more OPs expressing late markers O1 and RIP. Moreover, we discovered that iPS-derived OPs (iPS-OPs) engrafted 24 hours following a moderate contusive spinal cord injury (SCI) in rats survived for approximately two months and that more than 70% of the transplanted cells differentiated into mature oligodendrocytes that expressed myelin associated proteins. Transplanted OPs resulted in a significant increase in the number of myelinated axons in animals that received a transplantation 24 h after injury. In addition, nearly a 5-fold reduction in cavity size and reduced glial scarring was seen in iPS-treated groups compared to the control group, which was injected with heat-killed iPS-OPs. Although further investigation is needed to understand the mechanisms involved, these results provide evidence that patient-specific, iPS-derived OPs can survive for three months and improve behavioral assessment (BBB) after acute transplantation into SCI. This is significant as determining the time in which stem cells are injected after SCI may influence their survival and differentiation capacity.

Fig 1. Bright field microscopy images of the various stages of oligodendrocyte differentiation from induced pluripotent stem cells of the BC1 line.

(A) The undifferentiated cells grew in colonies on a mouse embryonic fibroblast feeder layer, (B) Embroid bodies (EBs) were formed by suspending the undifferentiating cells in culture on ultra-low adherence plates, (C) 15-day EBs were plated on matrigel, leading to the extension of cells of neuroectodermal lineage. Upon passaging, these neural progenitors grew in a monolayer and were subsequently differentiated to glial progenitors (D) stage. Addition of platelet derived growth factor-AA (PDGF-AA) to the media led to glial progenitors (GPs) transitioning into the (E) oligodendrocyte progenitor (OP) stage, displaying a rounded morphology with multiple filopodial extensions. (F) Terminal differentiation was induced by the removal of PDGF-AA, leading to the appearance of mature oligodendrocytes displaying characteristic branching. Scale bars- A-B: 300 μm, C-F: 80 μm

Fig 2. Plots showing mRNA expression of various developmental markers for all three iPS lines at each stage ofoligodendrocyte differentiation, as measured using qRT-PCR and normalized to fetal fibroblast controls.

Expression of pluripotency associated genes (A) NANOG, (B) Octamer-binding transcription factor 4 (OCT4) also known as POU5F1 (POU domain, class 5, transcription factor 1) and (C) SRY (sex determining region Y)-box 2 (SOX2) were undetected in oligodendrocyte progenitors (OPs) whilemarkers indicative of the oligodendrocyte lineage were induced including (D) NG2, (E) Myelin-associated glycoprotein (MAG), and (F) Myelin-associated oligodendrocytic basic protein (MOBP). Delta CTs were calculated using β-actin as a relative standard and lines normalized to fetal fibroblast controls. Mean + SEM were calculated from two independent differentiation experiments for each line and each gene performed in technical triplicate.Positive control included total RNA from human fetal brain. Abbreviations are Undiff: Undifferentiated iPS cells, EB: embryoid body, NP: neural progenitor, GP: glial progenitor, OP: oligodendrocyte progenitor.

Fig 3. Indirect immunofluorescence detection of neural markers help define the initial stages of oligodendrocyte differentiation from undifferentiated iPS cells, into neural (NP) and glial (GP) progenitors.

(A-D) Photos are of cells derived from BC1 with the differentiated stage (underlined) and marker analyzed as indicated similar staining patterns were seen in all three lines. Nuclei were stained using DAPI (blue). Most neural progenitors (NPs) were (A) NESTIN+ and (B) A2B5+ while the majority of glial progenitors (GPs) began expressing early oligodendrocyte progenitor (OP) markers (C) platelet derived growth factor receptor-α (PDGFR-α) and (D) NG2. Lower panel shows quantification of the indirect immunofluorescence analyses calculating the percent of labeled cells in all markers. (E) 90% of all cells from each line expressed early markers of the neural lineage including NESTIN and A2B5. (F) Likewise the number of glial progenitors expressing markers of the early OP lineage such as PDGFR-α and NG2 were also similar among lines. Differentiation of each cell line was performed in triplicate and the percent of positively stained cells was determined from 3 randomly, chosen 10X fields per 24-well cell culture dish from all three replicates. Values in the graph represent mean ± SEM. * and † denote statistical significance compared to BC1 and MR31, respectively by ANOVA and Tukey post hoc tests (N = 9, P < 0.001). Scale bars: 100 μm.

Fig 4. Indirect immunofluorescence marker characterization of oligodendrocyte progenitors derived from human iPS cells.

Nuclei were stained with DAPI (blue). Results show reduced expression of early markers (A) A2B5 and (B) NG2 and increased protein expression of mid to late oligodendrocyte progenitor (OP) differentiation markers (C) O4 (D) RIP (E) O1 (F) myelin-associated glycoprotein (MAG) and (G) myelin-associated oligodendrocytic basic protein (MOBP). Myelin basic protein (MBP) expression was not detectable by immunostaining. 1% or less of OP cells expressed markers of other neural lineages as denoted by (H) the neuronal maker neuron-specific class III beta-tubulin (TUJ1) and (I) the astrocyte marker glial fibrillary acidic protein (GFAP). (J) Quantification of the indirect immunofluorescence analyses calculating the percent of cells expressing a particular marker is shown in the lower panel. A higher percent of cells derived from BC1 expressed later OP markers MAG, MOBP and O1 while fewer cells from the BC1 lines expressed early neural progenitor (NP) and glial progenitor (GP) markers and early OP marker O4 compared to MR31 and A1-4. Differentiation of each cell line was performed in triplicate and the percent of positively stained cells was determined from 3 randomly, chosen 10X fields per 24-well cell culture dish from all three replicates. Values in the graphs represent mean ± SEM. * and † denote statistical significance compared to BC1 and MR31, respectively by ANOVA and Tukey post hoc tests (N = 9, P < 0.001). Scale bars: 100 μm.

Fig 5. Immunohistological characterization in rat spinal cords at the epicenter of injury (asterisks) following transplantation of either (A) heat-killed iPS-OPs or (B) live iPS-OPs, 24 hours after injury.

Human specific anti-nuclei antibody (HNA) staining showed that cells transplanted at the site of injury could survive, differentiate and express myelin basic protein (MBP, red) in iPS-OPs group, compared to the heat-killed iPS-OPs group. (C) Heat-killed iPS-OPs treated spinal cords demonstrated more cavitation and higher glial fibrillary acidic protein (GFAP) (green) expression than (D) spinal cords treated with iPS-OPs. Heat-killed iPS-OPs also expressed less MBP, a mature oligodendrocyte marker, than live iPS-OPs (red). (E) Double immunostaining with CD68 (expressed by macrophages, microglial, and T cells) and MBP shows more CD68+/MBP+ cells in control animals with morphology indicative of active macrophages or microglial engulfment of OPs. (F) Spinal cords injected with iPS-OPs had significantly fewer CD68+/MBP+ cells. Tissues shown here were isolated approximately 2 months after transplantation. DAPI was used to stain rat and human nuclei (blue). Scale bars for A, C, D: 100 μm and B, E, F: 30 μm. (G) Quantitative image analysis shows that more than 70% of HNA+ cells were differentiated MBP+ oligodendrocytes. Only 4.4 ± 1.5% of grafted cells express glial fibrillary acidic protein (GFAP), while no HNA+ cells were detected with co-localized staining for neuronal marker neuron-specific class III beta-tubulin (TUJ1) or NESTIN. Values represent mean ± SEM. * denote statistical significance of human MBP+ oligodendrocytes and MBP- OPs compared to either human-derived neural progenitors (Nestin+), neurons (TUJ1+) or astrocytes (GFAP+) and † denotes statistical significance of human HNA+MBP+ compared to HNA+MBP- OPs by Tukey post hoc tests. Tissue slices encompassing the injury epicenter were stained in intervals for each antibody combination from 4 rats in each treatment group, P < 0.001). (H) Optical density quantification of GFAP staining, which shows significantly higher intensity of GFAP staining in heat-killed iPS-OP compared with iPS-OPs of both BC1 and MR31 lines. (I) Quantification of the number of CD68+ cells shows significantly higher CD68+ cells in heat-killed iPS-OPs, indicative of increased inflammation and macrophage activation, compared with iPS-OPs of both BC1 and MR31 lines. Values in the graphs represent mean ± SEM. * and † denote statistical significance compared to heat-killed iPS-OPs and BC1-OPs, respectively by Tukey post hoc tests (10 identically numbered tissue slices encompassing the injury epicenter from 4 rats were analyzed for each treatment group, P < 0.001).

Fig 6. Hematoxylin and eosin and luxol fast blue stained paraffin sections of spinal cords from animals subjected to moderate contusive SCI and treated with (A) Heat-killed iPS-OPs or (B) Live, intact iPS-OPs derived from the BC1 line.

As expected for a 12.5 mm contusive injury, cavity formation was seen in both groups; however, the cavitation in spinal cords treated with iPS-OPs did not form well defined glial-like scar structures around the cavity periphery, compared to the control group. Thus, controls showed more pronounced cavitation, which extended further from the epicenter along the central canal. This indicates that the transplantation of live iPS-OPs into the newly formed cavity 24 hours after injury limited the normal expected progression of this level of contusive injury. The asterisks indicate the center of the injury site. Yellow boxes in A and B correspond to adjacent tissue sections stained in C and D, respectively. In these areas immediately surrounding the center of injury, staining with Luxol Fast Blue (LFB) to identify myelin showed reduced staining in the control group (C) versus the iPS-OP transplanted spinal cords (D). Thus, higher levels of LFB in the iPS-OP treated rats may indicate the presence of greater amounts of myelin. The representative spinal cords shown in (A-D) were harvested approximately 2 months after transplantation. Dotted lines help delineate regions of white matter (W) and grey matter (G). (E) Area measurements across groups revealed nearly a 5-fold significant reduction in cavity size in the iPS-OP treated groups compared to the heat-killed controls. Optical density of LBF, normalized to the optical density of the sham control group, was calculated for the grey matter (F) and white matter (G) of spinal sections to quantify the amount of myelin present in each area of the cord. Values in the graphs represent mean ± SEM. * denote statistical significance compared to heat-killed iPS-OPs by Tukey post hoc tests. For each stain, 5 identically numbered tissue slices encompassing the injury epicenter from 4 rats were analyzed for each treatment group, P < 0.001.

Fig 7. Transmission electron microscopy of transverse sections of the spinal cord showed remyelination of spared axons by BC1-derived iPS-OP cells after contusive SCI in rats.

(A) Normal, endogenous myelination of an uninjured rat spinal cord (laminectomy sham control) as indicated by white arrows, showing thick and tightly-packed myelin sheaths. (B) Remyelination observed in rats transplanted with BC1-derived OPs, approximately two months after transplantation. These are sites where oligodendrocytes are detected, as shown in Fig. 4. The relatively thinner and loosely packed myelin sheaths may be compared with the normal condition in (A), suggesting that this is remyelination by human OPs. (C) Multiple nude axons are visible in rats injected with heat-killed iPS-OPs, with no observable remyelination, approximately two months after transplant. (D) Quantification shows that transplantation of BC1-derived OPs resulted in a significant increase in the number of remyelinated axons and reduced demyelinated of axons compared with the heat-killed iPS-OP group. Values in the graphs represent mean ± SEM. * denote statistical significance compared to heat-killed iPS-OPs by Tukey post hoc tests. Ten images encompassing the injury epicenter from 4 rats were analyzed for each treatment group (P < 0.001). Magnification (A-C): 10000X.

Fig 8. Behavioral assessment 8 weeks following moderate contusive injury (12.5mm) and cell transplantation in rats.

The average of the Basso, Beattie, and Bresnahan (BBB) Locomotor Rating Scale (BBB SCORE) for left and right limb movement was evaluated for 55 days following spinal cord injury in rats injected with either PBS (blue), 500,000 heat-killed iPS-OP cells (green), live BC1-OP (black), or MR31-OP (red) cells 24 hrs after injury. Values in the graphs represent mean ± SEM of both left and right limbs as there were no significant differences in response between right and left limb scores. Statistical significance is denoted by a colored asterisk for each cell line with matched color compared to the PBS control group determined by one-way ANOVA followed by a two-way Student’s t-test assuming equal variances (P < 0.025).