Mechanosensitivity of Human Oligodendrocytes

Abstract

Oligodendrocytes produce and repair myelin, which is critical for the integrity and function of the central nervous system (CNS). Oligodendrocyte and oligodendrocyte progenitor cell (OPC) biology is modulated in vitro by mechanical cues within the magnitudes observed in vivo. In some cases, these cues are sufficient to accelerate or inhibit terminal differentiation of murine oligodendrocyte progenitors. However, our understanding of oligodendrocyte lineage mechanobiology has been restricted primarily to animal models to date, due to the inaccessibility and challenges of human oligodendrocyte cell culture. Here, we probe the mechanosensitivity of human oligodendrocyte lineage cells derived from human induced pluripotent stem cells. We target phenotypically distinct stages of the human oligodendrocyte lineage and quantify the effect of substratum stiffness on cell migration and differentiation, within the range documented in vivo. We find that human oligodendrocyte lineage cells exhibit mechanosensitive migration and differentiation. Further, we identify two patterns of human donor line-dependent mechanosensitive differentiation. Our findings illustrate the variation among human oligodendrocyte responses, otherwise not captured by animal models, that are important for translational research. Moreover, these findings highlight the importance of studying glia under conditions that better approximate in vivo mechanical cues. Despite significant progress in human oligodendrocyte derivation methodology, the extended duration, low yield, and low selectivity of human-induced pluripotent stem cell-derived oligodendrocyte protocols significantly limit the scale-up and implementation of these cells and protocols for in vivo and in vitro applications. We propose that mechanical modulation, in combination with traditional soluble and insoluble factors, provides a key avenue to address these challenges in cell production and in vitro analysis.

Keywords: glia; human models; in vitro; induced pluripotent stem cell; mechanobiology; mechanotransduction; oligodendrocyte.

Figure 1

Differentiation of human oligodendrocytes from human induced pluripotent stem cells (hiPSCs) obtained from healthy donors, on compliant polyacrylamide (PAAm) hydrogels. (A) Human oligodendrocytes were differentiated as previously reported (Douvaras et al., 2014). At day 30 of the differentiation protocol, spheroids were plated on polyacrylamide hydrogels and tissue culture polystyrene functionalized with poly-L-ornithine and laminin. After 30 days (day 60), oligodendrocyte lineage cells were recognized by the transcription factor SOX10 (green). Migration was measured as the total surface area of the hydrogel covered by cells outside of the spheroid. Cells were grown for an additional 8 days (day 68) in maturation medium, and oligodendrocytes were stained with the O4 antibody (pink). (B) Range of stiffness (Young’s modulus) reported for brain tissue and typical oligodendrocyte cell culture substrata in the literature (Makhija et al., 2020). (C) PAAm hydrogels were prepared to span the range of physiological and pathological brain stiffness previously probed with rat primary oligodendrocytes (Jagielska et al., 2012). Mean ± standard deviation; n = 3 hydrogel samples for each material stiffness.

Figure 2

Human oligodendrocyte lineage cell migration is inhibited on compliant substrata, over the range of elastic moduli E = 0.1–70 kPa. (A) Representative images depicting spheroids (bright region in image center) and area of migration of DAPI-stained cells away from spheroids generated from donor cell line 2. Scale bars are 1 mm. (B) Propensity for migration of all nucleated DAPI-stained cells correlated positively with increasing stiffness of polyacrylamide hydrogel substrata, over the range of E = 0.1–70 kPa, for all donor cell lines. Values are mean ± standard deviation; n ≥ 2 spheroids per hydrogel substratum stiffness. (A,B) Migration from the spheroids was quantified as the total area enclosed by the outer perimeter of cells further away from the spheroid, and an inner perimeter demarcated by the edge of the spheroid. Spearman correlation coefficient was calculated across hydrogels. All coefficients were statistically significant, with p < 0.05. (C) The propensity for migration of oligodendrocyte lineage cells (SOX10+ cells) was comparable to that of the entire population (DAPI); r is the Pearson correlation coefficient; n = 22 spheres; pooled from two independent experiments. This can be visually appreciated in Supplementary Figure S1A. (D) Comparison of migration on compliant polyacrylamide (PAAm) hydrogels and TCP. (E) Comparison of migration on stiff polyacrylamide (PAAm) hydrogels and tissue culture polystyrene (TCP). (D,E) Migration was quantified as in (B). Values are mean ± standard deviation; points are spheroids pooled from two independent experiments per line. Hydrogel data points are the same as in panel (B). Two-tailed t-test, *p < 0.05, **p < 0.005 and ***p < 0.0005 for hydrogels compared with their respective TCP controls. Supplementary Figure S11 contains the graphs from panel (B) with adjusted axes to facilitate visual appreciation of trends across hydrogel substrata.

Figure 3

Human oligodendrocyte differentiation is modulated by substratum stiffness. (A) Representative images of O4+ cells (magenta) generated from two donor lines (2 and 5) that exhibited opposite mechanosensitive differentiation trends. DAPI-stained nuclei are shown in green. Scale bars are 200 μm. (B) Magnified image of O4+ cells. (C) Oligodendrocyte fraction (defined as percentage of O4+ cells) varied repeatably for a given donor with substratum stiffness, over the range of apparent Young’s elastic modulus E = 0.1–70 kPa. The percentage of O4+ cells was quantified as the number of O4+ cells with DAPI-stained nuclei, relative to the total number of DAPI-stained cells. However, the direction of the correlation (positive or negative correlation with stiffness) was donor dependent. Specifically, hiPSC donor lines 2 and 4 exhibited increased differentiation (% O4+ cells) with increasing material stiffness (type B response), whereas lines 3 and 5 showed decreased percentage of O4+ cells with increasing hydrogel stiffness (type A response). Data shown as mean ± standard deviation; n = 2–4 spheroids per stiffness condition. Spearman correlation coefficient was calculated across polyacrylamide hydrogels, and all coefficients were statistically significant with p < 0.05. Supplementary Figure S11 contains the graphs from panel (C) with adjusted axes to facilitate visual appreciation of trends across hydrogel substrata.

Figure 4

Schematic representation of findings. Human iPSC-derived oligodendroglia (OL) migrated further away from the edge of the spheroids when grown on stiffer hydrogel substrata (apparent Young’s elastic modulus or stiffness, E = 0.1–70 kPa), consistent for all donor lines studied. Some hiPSC donor lines, identified as type A, exhibited diminished differentiation, or lower fraction of O4+ cells, with increasing substratum stiffness. Conversely, type B lines exhibited enhanced differentiation r greater fraction of O4+ cells with increasing substratum stiffness.