A cord blood monocyte-derived cell therapy product accelerates brain remyelination

Abstract

Microglia and monocytes play important roles in regulating brain remyelination. We developed DUOC-01, a cell therapy product intended for treatment of demyelinating diseases, from banked human umbilical cord blood (CB) mononuclear cells. Immunodepletion and selection studies demonstrated that DUOC-01 cells are derived from CB CD14+ monocytes. We compared the ability of freshly isolated CB CD14+ monocytes and DUOC-01 cells to accelerate remyelination of the brains of NOD/SCID/IL2Rγnull mice following cuprizone feeding-mediated demyelination. The corpus callosum of mice intracranially injected with DUOC-01 showed enhanced myelination, a higher proportion of fully myelinated axons, decreased gliosis and cellular infiltration, and more proliferating oligodendrocyte lineage cells than those of mice receiving excipient. Uncultured CB CD14+ monocytes also accelerated remyelination, but to a significantly lesser extent than DUOC-01 cells. Microarray analysis, quantitative PCR studies, Western blotting, and flow cytometry demonstrated that expression of factors that promote remyelination including PDGF-AA, stem cell factor, IGF1, MMP9, MMP12, and triggering receptor expressed on myeloid cells 2 were upregulated in DUOC-01 compared to CB CD14+ monocytes. Collectively, our results show that DUOC-01 accelerates brain remyelination by multiple mechanisms and could be beneficial in treating demyelinating conditions.

Figures

Figure 1. Severe demyelination of the midline corpus callosum (CC) area and glial infiltration of NSG mouse brain by cuprizone feeding.

(A) LFB-PAS staining of NSG mice brain after 5 weeks of feeding with (right panel) and without (left panel) 0.2% cuprizone (CPZ). The midline CC area is shown by dotted red boxes in the top panels and then shown at higher magnification in the lower panels. Myelinated axons in the CC of mice fed normal laboratory chow are stained blue. Demyelination of the midline CC region of CPZ-treated animals is shown by the absence of the blue-colored fibers. Scale bars: 2,000 μm (×20 magnification) and 100 μm (×400 magnification). (B) Myelin basic protein immunostaining (green) after 5 weeks of feeding without (left panel) and with CPZ (right panel). Two different magnifications (top row is ×100 and bottom row is ×400) of the CC areas are shown. CC areas are shown by white dotted lines. (C) Immunostaining with microglial marker Iba1 (red, upper panels) and astrocyte marker GFAP (pink, lower panels) after 5 weeks of feeding without (left panels) and with CPZ (right panels). CC areas are shown by white dotted lines. Scale bars: 200 μm. (D) Quantitative analysis of area covered by Iba1-positive (upper panel) and GFAP-positive (lower panel) cells, indicative of their numbers, along the CC. Both Iba1-positive and GFAP-positive cell numbers were significantly higher in the CPZ-treated animals. *P < 0.02, **P < 0.004. n = 3 mice per group . C, control. Data are presented as the mean ± SEM.

Figure 2. Some DUOC-01 cells disseminated from the injection site and persisted in the brain for up to 1 week after intracranial injection.

Cuprizone-fed (CPZ-fed) mice were stereotactically injected with CFSE-labeled DUOC-01 cells. All cell nuclei were stained with DAPI (blue). (A) CFSE-labeled (green) DUOC-01 cells were found in numerous parts of the brain including the injection site. Scale bars: 200 μm. CC, corpus callosum; SV, subventricular. (B) Representative images of CFSE-positive (green) and human nuclei (HuN, red)-positive cells in the brain at the injection site 4 days after injection. Upper left panel is CFSE (green) channel only, lower left panel is HuN (red) channel only, right panel is merge of CFSE, HuN, and DAPI channels. (C) Upper left panel is CFSE channel only, lower left panel is HuN channel only, right panel is merge of CFSE, HuN, and DAPI channels showing presence of DUOC-01 cells 7 days after injection at the CC. (D) Upper left panel is CFSE channel only, lower left panel is HuN channel only, right panel is merge of CFSE, HuN, and DAPI channels showing presence of DUOC-01 cells deep (white arrow) into the brain parenchyma. Scale bars (B–D): 100 μm.

Figure 3. LFB-PAS staining analysis of effect of DUOC-01 treatment on remyelination following cessation of cuprizone (CPZ) treatment.

(A) LFB-PAS staining 1 week after intracranial injection of CD14+ monocytes (lower panels), DUOC-1 cells (middle panels), or Ringer’s solution (upper panels) in CPZ-fed NSG mice. Midline corpus callosum (CC) area is shown by dotted green line. Scale bars: 2,000 μm (×20 magnification) and 100 μm (×400 magnification). (B) Myelination score based on LFB-PAS staining of mice fed normal chow (control) or CPZ for 5 weeks 1 week after treatment of CPZ-treated mice with CD14+ monocytes, DUOC-01 cells, or Ringer’s. DUOC-01 treatment for 1 week significantly increased the myelination in the CC area compared to Ringer’s-injected controls. **P < 5.962 × 10–5 for this study. The CD14+ cell–treated sample showed an increased amount of remyelination compared to the Ringer’s-treated group, but it was significantly less than the DUOC-01–treated group. *P < 0.003875. Data are presented as the mean ± SEM. Statistical comparisons were performed using the Wilcoxon rank-sum test for clustered data using the clusrank package in R.

Figure 4. Immunostaining analysis of the effect of DUOC-01 treatment on remyelination following cessation of cuprizone (CPZ) treatment.

In all images, myelin basic protein (MBP) staining is shown in green. (A) Representative ×400 laser confocal images of sections of the corpus callosum (CC) area of CPZ-fed mice immunostained with antibodies against MBP (green) and neurofilament-H (NFH, purple), 1 week after treatment with Ringer’s solution (A), DUOC-01 (B), or CD14+ (C). Upper left panels show MBP (green channel), lower left panels show NFH (purple channel), and right panels show enlarged merge images of MBP and NFH channels. Scale bars: 100 μm.

Figure 5. Electron microscopic analysis of remyelination status upon DUOC-01 treatment.

Representative ×2,650 (upper panels) and ×8,800 (lower panels) electron micrographs of corpus callosum region of cuprizone-fed mice 1 week after injection of Ringer’s solution (left panels) or DUOC-01 cells (right panels). Blue arrows indicate unmyelinated axons. Red arrowheads indicate mitochondria; enlarged mitochondria are clearly visible in the Ringer’s-treated group. Scale bars: 2.0 μm.

Figure 6. Morphometric analysis of electron micrographs of corpus callosum regions of DUOC-01– and Ringer’s-treated mice.

(A) Number of myelinated axons present per ×8,800 electron microscopy field. Data are presented as the mean ± SEM showing all the data points. *P ≤ 4.29 × 10–9. (B) Average number of turns of myelin sheath around axons, with right panels showing a representative electron micrograph of the myelin turns in an axon. *P ≤ 3.4 × 10–6. Scale bars: 100 nm. (C) Scatter plot of g-ratios, showing axonal measurements from 3 different animals in each group. Horizontal lines indicate mean g-ratios. P ≤ 0.014. (D) Average size of mitochondria (area in nm2). Mean difference is significant between DUOC-01 and Ringer’s groups. *P ≤ 9.3 × 10–5. (E) Average number of mitochondria per ×8,800 field. The mean difference is significant between DUOC-01 and Ringer’s groups. *P ≤ 0.02. Each column represents the value of measurements from 3 different animals. Error bars indicate the SEM. Statistical comparisons were performed using an unpaired 2-tailed Student’s t test.

Figure 7. DUOC-01 cell treatment reduces severe astrogliosis and microglial infiltration.

(A) A quantitative cellularity scoring of LFB-stained brain slices on a scale of 0 to 3. **P ≤ 7.618 × 10–5, n ≥ 5. Control, not cuprizone fed; CPZ, cuprizone fed; Ringer’s, 1 week after Ringer’s injection; DUOC-01, 1 week after DUOC-01 injection. Data are presented as the mean ± SEM showing each data point. Statistical comparisons were performed using the Wilcoxon rank-sum test for clustered data using the clusrank package in R. (B) Cellularity status by immunostaining using astrocyte-specific (GFAP, right panels) and microglia-specific (Iba1, left panels) markers. Midline corpus callosum (CC) areas are shown in dotted line. Scale bars: 100 μm. (C) Quantitative analysis of area covered by Iba1-positive (upper panel) and GFAP-positive (lower panel) cells, indicative of their numbers, along the CC. Both the numbers of Iba1-positive (microglia) and GFAP-positive (astrocytes) cells were significantly lower in the DUOC-01–treated mice. *P < 0.002; **P < 0.01. n = 3 mice per group. Areas covered by each channel (either GFAP or Iba1) per microscopic field were quantified by ImageJ software. Data are presented as the mean ± SEM. Statistical comparisons were performed using an unpaired 2-tailed Student’s t test.

Figure 8. DUOC-01 treatment promotes oligodendrocyte proliferation.

(A) Representative image of corpus callosum area of brains of cuprizone-fed mice treated with DUOC-01 cells (lower panels) or Ringer’s solution (upper panels) stained with antibodies against Olig2 and Ki67. Yellow arrows indicate nuclei positive for both Olig2 and Ki67, blue arrows indicate only Ki67-positive nuclei. Scale bars: 50 μm. (B) Average number of Olig2+Ki67+ cells (indicating proliferating oligodendrocytes) present per ×400 microscopic field were significantly higher in DUOC-01–treated samples compared to the Ringer’s control. *P < 0.01. Statistical comparisons were performed using an unpaired 2-tailed Student’s t test.

Figure 9. Comparative whole-transcriptome analysis of CD14 and DUOC-01 cells.

(A) Venn diagram displaying the findings from microarray analysis showing the number of genes differentially expressed in purified fresh CD14+ (n = 4) and DUOC-01 (n = 3) cells as well as genes expressed by both cell types. MAS5-normalized data were used to filter out expressed/nonexpressed genes. The figure represents the most stringent analysis; to be scored as expressed, the transcript had to be detected above background in all samples of a given cell type analyzed. See the text for expression figures at different stringencies. (B) Volcano plot depiction of findings from microarray analysis showing the genes differentially expressed in purified fresh CD14+ and DUOC-01 cells. The log10 of Bonferroni-Hochberg–corrected P values in ANOVA (y axis) is plotted against the of fold change between 2 groups (x axis). Red lines delineate the cutoffs for genes significantly (P < 0.05) downregulated (left) or upregulated (right) in DUOC-01 cells. Each data point represents 1 gene probe set. (C) Heat maps showing differentially expressed genes. Up- and downregulated genes are displayed in red and blue, respectively. There were 9,645 genes that are differentially expressed at a magnitude of at least 2-fold.