1. Volume 159, Issue 4, Pages 766-774 (November 2014)
Dynamics of Oligodendrocyte Generation and Myelination in the Human Brain 
Maggie S.Y. Yeung, Sofia Zdunek, Olaf Bergmann, Samuel Bernard, Mehran Salehpour, Kanar Alkass, Shira Perl, John Tisdale, Göran Possnert, Lou Brundin, Henrik Druid, Jonas Frisén 
Cell 
Volume 159, Issue 4, Pages (November 2014)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Oligodendrocyte Generation in the Human Corpus Callosum
(A) Oligodendrocyte progenitor cells (OPC, SOX10+, NOGO-A−, arrows) and mature oligodendrocytes (SOX10+/NOGO-A+, solid arrowheads) were identified in histological sections from the human postmortem corpus callosum and quantified by stereology. Immature oligodendrocyte lineage cells lack NOGO-A and nonoligodendrocyte lineage cells also lack SOX10 (hollow arrowheads). Cell nuclei are visualized with TO-PRO-3. Scale bar represents 10 μm.
(B and C) The number of oligodendrocyte progenitor cells (B) and oligodendrocytes (C) in the human corpus callosum. Males are indicated with blue circles and females with red circles. The solid lines represents a double exponential fitting of the data (see also Extended Experimental Procedures) and the dashed lines represent 95% confidence bands.
See also Figure S1 and Tables S1 and S2.
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4. Figure 2 Myelination of the Human Corpus Callosum
(A) The myelin volume in the corpus callosum of the same subjects as in Figures 1B and 1C. The solid line represent double exponential curve fitting and the dashed lines represent 95% confidence bands.
(B) There is no correlation between the number of oligodendrocytes and the myelin volume in the corpus callosum (Pearson’s correlation, r = −0.10, p = 0.52) once the final complement of oligodendrocytes is established. Data for individuals >5 years of age are shown. Males are indicated in blue and females in red.
See also Figure S2 and Tables S3 and S4.
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5. Figure 3 Myelin Is Young and White Matter Cells Are Old
(A) Schematic depiction of the presentation of 14C data. The curve indicates the atmospheric 14C concentration over time. The data (circles) is plotted based on the date of birth (DoB) of the person and the 14C concentration in myelin or genomic DNA. For individuals born after the nuclear bomb tests (1955–1963), the date of generation (DoG) of cells or myelin can be inferred by reading of the x axis, whereas this cannot be directly inferred for subjects born before the onset of the increase in 14C. If not otherwise stated, the date of death of the studied subjects was 2009–2012 in all figures.
(B) The 14C concentration in genomic DNA from corpus callosum (black circles) and frontal lobe white matter (white diamonds) cells corresponds to within a few years after the birth of individuals born after the nuclear bomb tests. In subjects born before the onset of the nuclear bomb tests, the 14C concentration is lower than contemporary levels, indicating that a substantial proportion of white matter cells have not been exchanged for at least 5 decades.
(C) The 14C concentration in biochemically isolated myelin from the corpus callosum corresponds to the time around the death (indicated with arrows and dashed line to the respective data points) of the subjects, demonstrating that myelin is contemporary and is exchanged at a high rate. Error bars in (B) and (C) indicate 2 SD.
See also Table S5.
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6. Figure 4 Isolation of Oligodendrocyte Nuclei
(A) Isolation of oligodendrocyte nuclei from the human postmortem corpus callosum by flow cytometry. Labeling with isotype control antibodies (left) and SOX10 and APC antibodies (right) are shown.
(B) Quantitative RT-PCR reveals that almost all of the mRNA for SOX10 as well as the mature oligodendrocyte markers myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG) is present in the SOX10+/APC+ nuclei (encircled by red hatched line in [A] and represented by red bar in [B]) and only little of the mRNA is found in the nonoligodendrocyte fraction (marked in orange in [A] and [B]). Data in (B) represent the average from three independent experiments, mean ± SD (n = 3 individuals).
See also Figure S3.
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7. Figure 5 Birth Dating Oligodendrocytes
14C concentrations in genomic DNA of SOX10+/APC+ oligodendrocytes demonstrate limited cell turnover in subjects who died 2009–2012 (A) and in archival specimen from individuals (1-6, arrows below the 14C curve) with earlier death dates (date of death, 1-6, arrows above the 14C curve) (B). Males are indicated in blue and females in red. Error bars show 2 SD.
See also Figure S4 and Table S5.
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8. Figure 6 Oligodendrocyte Turnover Dynamics in Humans
(A) Mathematical modeling reveals that the 14C incorporation in oligodendrocyte genomic DNA is best explained by a stable turnover rate of 0.32% per year after 5 years of age, without any substantial increase in cell number. 14C data points are shown as blue circles, values deduced by the model are depicted by red line.
(B) With the limited exchange of oligodendrocytes, the average age of this population is only a few years younger than the subject. The hatched line indicates the no turnover scenario.
See also Figure S5 and Table S6.
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9. Figure 7 Time Line for Oligodendrocyte Generation in Humans
(A) There is no correlation between the turnover of oligodendrocytes and the oligodendrocyte number, and the data exclude any major change in oligodendrocyte number after 5 years of age (indicated with red hatched lines for a subset of data points). The number of oligodendrocytes is determined ∼5 years of age and stays constant thereafter (green lines).
(B) Representation of the population of oligodendrocytes that have been generated until 5 years of age (gray) and the proportion that is exchanged with time (blue). The figure is based on the 14C data, but constrained by the stereological data to also be in accordance with the cell number development, see Extended Experimental Procedures.
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10. Figure S1
Oligodendrocyte Identification in the Human Corpus Callosum, Related to Figure 1
(A) Mature oligodendrocytes (SOX10+/NOGO-A+/APC+, arrowheads) in histological section from the human postmortem corpus callosum. An immature oligodendrocyte lineage cell expresses SOX10, but lacks the mature markers NOGO-A and APC (arrows). Nonoligodendrocyte lineage cells in addition lack SOX10 (hollow arrowheads).
(B) Mature oligodendrocytes (SOX10+/APC+, arrowheads) also express myelin basic protein (MBP). An immature oligodendrocyte lineage cell lack APC and MBP (arrow) and a nonoligodendrocyte lineage cell also lacks SOX10 (hollow arrowhead).
(C and D) Nonoligodendrocyte lineage cells were identified as microglia (Iba+, hollow arrowhead in (C) or astrocytes (GFAP+, hollow arrowheads in (D). IBA1+ microglia constitute 7.1% and GFAP+ astrocytes account for 4.3% of the cells in the corpus callosum. Mature oligodendrocytes (SOX10+/APC+, arrowheads) and immature oligodendrocyte lineage cells as (SOX10+ only, arrow) are indicated. Cell nuclei are visualized with TO-PRO-3 or DAPI. The scale bars are 10 μm.
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11. Figure S2
The Myelin Volume Parallels the Volume of the Corpus Callosum, Related to Figure 2
(A) Validation of myelin volume measurement by increasing or decreasing ratio of more myelinated corpus callosum tissue versus less myelinated cerebellar tissue. Each data point represents the average from three independent experiments (mean ± SD, n = 3 subjects).
(B) The myelin volume in the human corpus callosum parallels the total volume of corpus callosum established by imaging. The gray data and solid line represents a Poisson curve fitting of the data of total volume of corpus callosum (Lebel et al., 2012), and the black solid line represent double exponential curve fitting of the measured myelin volume data. The dashed lines represent 95% confidence bands.
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12. Figure S3
Reanalysis of Flow Cytometry Isolated Oligodendrocyte and Nonoligodendrocyte Nuclei and Quantitative RT-PCR Analysis, Related to Figure 4
(A) Reanalysis of flow cytometry isolated oligodendrocyte nuclei show 1.8% ± 1.2% contamination of nonoligodendrocyte nuclei in the SOX10+/APC+ population.
(B) Reanalysis of flow cytometry isolated nonoligodendrocyte nuclei show 2.4% ± 1.6% contamination of oligodendrocyte nuclei. A minimum of 5,000 nuclei was reanalyzed in all 14C measured samples. Data in (A) and (B) represent mean ± SD, n = 34 subjects.
(C) Quantitative RT-PCR reveals that most of the mRNA for nonoligodendrocyte markers such as the astrocytic marker GFAP, hematopoietic and microglial marker CD45 and immature oligodendrocyte cell linage marker NG2 is present in the nonoligodendrocyte nuclei fraction (orange), with much less present in the SOX10+/APC+ fraction (red). mRNA for endothelial marker VWF was not detectable and therefore not shown. Data in (C) represent mean ± SD (n = 3 individuals and independent experiments)
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13. Figure S4
Assessment of Oligodendrocyte Turnover, Related to Figure 5 and Table S5
(A) 14C levels in genomic DNA of frontal white matter SOX10+/APC+ oligodendrocyte nuclei demonstrate similar 14C levels as in oligodendrocyte nuclei isolated from the corpus callosum.
(B) Isolated cortical and cerebellar neurons (NeuN+) from the same subject showed 14C levels corresponding to the subject’s date of birth. Error bars in (A) and (B) indicate 2 SD.
(C and D) 14C concentrations in genomic DNA of SOX10+/APC+ oligodendrocytes in prefrontal or frontal cortex gray matter demonstrate higher cell turnover compared to white matter oligodendrocytes in (C) same subjects who died and (D) in archival specimen from individuals (1-6, arrows below the 14C curve) with earlier death dates (date of death, 1-6, arrows above the 14C curve). Males are indicated in blue and females in red. Error bars show 2 SD.
(E and F) Frequency of proliferating cells (Ki67+) and proliferating oligodendrocyte progenitor cells (Ki67+/SOX10+/NOGO-A-, hollow arrowheads) were abundant in histological sections from the human postmortem corpus callosum from subject aged 0.4 years (E), compared with subject aged 4.7 years (F). Dashed box indicate area of orthogonal view. Proliferating cells of nonoligodendrocyte lineage (Ki67+ only, arrowheads) are indicated.
(G) Quantification of Ki67+ cells in the human corpus callosum. Data represent mean ± SD, n = 3 sections/subject.
(H) Only one IdU-labeled cell expressing the mature oligodendrocyte marker NOGO-A (hollow arrowhead) was found (among analysis of > mature oligodendrocytes in each patient, n = 3 patients) in histological section from human postmortem cortex, from subjects who received the thymidine analog IdU as a radiosensitizer. A non-IdU incorporated mature oligodendrocyte (NOGO-A+, arrow) is indicated. Cell nuclei are visualized with DAPI or histone H3. The scale bars are 20 μm.
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14. Figure S5
Individual Turnover Rate of Oligodendrocytes in the Corpus Callosum, Related to Figure 6
(A) Mathematical modeling of the 14C data with different plateau times (1-17 years) show that the best fit is obtained for years 3 to 5. A blue circle represents the goodness-of-fit value obtained when fitting the 14C data with plateau time as indicated on the x axis. The red line indicates the interpolated values.
(B) Mathematical modeling of the individual turnover rate of oligodendrocytes shows no correlation with age (Pearson’s correlation: r = 0.055, p = 0.76). Negative turnover rates indicate that the individual cell number expansion, during the first 5 years, is less than the fitted cell number expansion for all subjects together.
See also Extended Experimental Procedures.
Cell  , DOI: ( /j.cell )
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