Cell Replacement Therapy Improves Pathological Hallmarks in a Mouse Model of Leukodystrophy Vanishing White Matter

Summary: Stem cell therapy has great prospects for brain white matter disorders, including the genetically determined disorders called leukodystrophies. We focus on the devastating leukodystrophy vanishing white matter (VWM). Patients with VWM show severe disability and early death, and treatment options are lacking. Previous studies showed successful cell replacement therapy in rodent models for myelin defects. However, proof-of-concept studies of allogeneic cell replacement in models representative of human leukodystrophies are lacking. We tested cell replacement in a mouse model representative of VWM. We transplanted different murine glial progenitor cell populations and showed improved pathological hallmarks and motor function. Improved mice showed a higher percentage of transplanted cells that differentiated into GFAP+ astrocytes, suggesting best therapeutic prospects for replacement of astroglial lineage cells. This is a proof-of-concept study for cell transplantation in VWM and suggests that glial cell replacement therapy is a promising therapeutic strategy for leukodystrophy patients. : Vanishing white matter (VWM) is a severe genetic white matter disorder for which no curative treatment is available. Heine and colleagues show that transplantation with glial progenitor cells can improve pathology and motor skills in VWM mice. Improvement was correlated with increased astrocyte differentiation of donor cells, showing that astrocyte targeting is essential for VWM therapy. Keywords: cell replacement therapy, astrocytes, oligodendrocytes, glial cells, white matter disorder, vanishing white matter, leukodystrophy, stem cell

Astrocyte Subtype Vulnerability in Stem Cell Models of Vanishing White Matter

Objective: Astrocytes have gained attention as important players in neurological disease. In line with their heterogeneous character, defects in specific astrocyte subtypes have been identified. Leukodystrophy vanishing white matter (VWM) shows selective vulnerability in white matter astrocytes, but the underlying mechanisms remain unclear. Induced pluripotent stem cell technology is being extensively explored in studies of pathophysiology and regenerative medicine. However, models for distinct astrocyte subtypes for VWM are lacking, thereby hampering identification of disease-specific pathways. Methods: Here, we characterize human and mouse pluripotent stem cell–derived gray and white matter astrocyte subtypes to generate an in vitro VWM model. We examined morphology and functionality, and used coculture methods, high-content microscopy, and RNA sequencing to study VWM cultures. Results: We found intrinsic vulnerability in specific astrocyte subpopulations in VWM. When comparing VWM and control cultures, white matter–like astrocytes inhibited oligodendrocyte maturation, and showed affected pathways in both human and mouse cultures, involving the immune system and extracellular matrix. Interestingly, human white matter–like astrocytes presented additional, human-specific disease mechanisms, such as neuronal and mitochondrial functioning. Interpretation: Astrocyte subtype cultures revealed disease-specific pathways in VWM. Cross-validation of human- and mouse-derived protocols identified human-specific disease aspects. This study provides new insights into VWM disease mechanisms, which helps the development of in vivo regenerative applications, and we further present strategies to study astrocyte subtype vulnerability in neurological disease. ANN NEUROL 2019;86:780–792

Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells

<div><p>Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory-inhibitory activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons. We used two different co-culture models with astrocytes. We show that these cultures have balanced excitatory-inhibitory synaptic identities using confocal microscopy, electrophysiological recordings, calcium imaging and mRNA analysis. These simple and robust protocols offer the opportunity for single-cell to multi-level analysis of patient hiPSC-derived cortical excitatory-inhibitory networks; thereby creating advanced tools to study disease mechanisms underlying neurodevelopmental disorders.</p></div

Characterization of the differentiated neurons in indirect contact co-cultures.

<p>In indirect co-cultures we differentiated the human neurons in close proximity of rat astrocyte layers (Line B1 and B2). (A, B) Bright field images of indirect cultures during differentiation day 26 and 56 are shown. (C-F) We observed the expression of MAP2, VGAT, synaptophysin1 and (G-J) VGLUT1 and HOMER1 co-localizing puncta using immunocytochemistry at day 56. We then analyzed (K) spontaneous mEPSCs and mIPSCs (holding potential at -70mV in voltage clamp mode) recorded in 1 μM TTX supplemented with 40 μM Bicuculline or 50 μM AP5 + 10 μM DNQX respectively. (L) Quantification of amplitude, area, time to decay and frequency of mEPSCs and mIPSCs was also analyzed (n = 2). We then recorded calcium traces of (M, N) neurons loaded with Fluo-5 AM ester from n = 2. (O) Raster plot showing onset and duration of intracellular calcium events from ROIs represented on M and N respectively; upon TTX addition and 20 minutes after TTX was washed away (P) or upon bicuculline, bicuculline + AP5 + CNQX addition and 20 minutes after drugs were washed away (representative calcium traces for ROI 1(M, O) and ROI 8 (N, P)) are shown. (Q) Analysis of activity dependent intracellular calcium traces (red dotted bars indicate beginning of electrical field stimulation and number of pulses applied). Scale bars are 10 μm. Data are represented as mean ± SEM. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178533#pone.0178533.s005" target="_blank">S5 Fig</a> for indirect contact plate preparation method.</p

Functional analysis of differentiated neurons in direct contact co-cultures.

<p>At day 49 we recorded Line A-derived (n = 2) and Line B-derived (n = 2) neurons, which show (A) fast inward Na⁺ currents followed by long-lasting outward K⁺ currents; current was evoked by voltage steps ranging from -90 mV to 50 mV with 10 mV increments. Inset shows fast Na⁺ currents in absence or presence of Na⁺ channel antagonist TTX (1 μM). (B) Quantification of peak Na⁺ and K⁺ currents evoked in (A) is shown. (C) We recorded action potentials evoked by a current injection of 100 pA and also (D) spontaneously generated action potentials in current clamp mode. (E) Spontaneous post-synaptic events from a single neuron (holding potential at -70 mV in voltage clamp mode) recorded in presence of 1μM TTX, TTX + 40 μM Bicuculline and TTX + 40 μM Bicuculline + 50 μM AP5 + 10 μM DNQX are shown. (F) Postsynaptic current evoked by local field stimulation (2 mA, 1 ms). We recorded calcium traces at day 49 of differentiation; (G, H) hiPSC-derived neurons loaded with Fluo-5 AM ester. (I) Raster plot showing onset and duration of intracellular calcium events from ROIs represented on G; upon TTX addition and 20 minutes after TTX was washed away (J) or traces represented in H upon bicuculline, bicuculline + AP5 + CNQX addition and 20 minutes after drugs were washed away (representative calcium traces for ROI 2 (G, I) and ROI 9 (H, J)) are shown. (K) We then analyzed activity dependent intracellular calcium traces (red dotted bars indicate beginning of electrical field stimulation and number of pulses applied). Scale bars are 10 μm. Data are represented as mean ± SEM. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178533#pone.0178533.s004" target="_blank">S4 Fig</a> for hESC-derived neurons calcium imaging experiments.</p

hNES cell-derived neuronal culture protocol.

<p>hiPSC-derived NES cells were generated and characterized by (A-E) immunocytochemistry for PLZF, PAX6, SOX2, (F) morphology as well as (G) RT-PCR for <i>SOX2</i>, <i>Nestin</i>, <i>PAX6</i>, <i>PLZF</i>, <i>DACH1</i>, <i>ZNF312</i> and <i>HES5</i>, and later differentiated into low-density neuronal cultures. (H) Schematic representation of the differentiation protocol. (I-L) Representative images of differentiating neurons at day 4, 8, 20 and direct contact culture mode day 49 are shown. (M, N) Cartoon representation of direct and indirect contact co-culture model. Scale bars are 25 μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178533#pone.0178533.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178533#pone.0178533.s002" target="_blank">S2</a> Figs for hiPSC characterization and hESC-derived hNES cell characterization.</p

Proteomic analysis of indirect contact co-culture neurons representing reproducible cultures.

<p>(A) Hierarchical clustering of protein groups based on expression intensity present in the day 56 differentiated neurons. Columns represent triplicates from 2 experiments (B1 and B2) normalized for loading and appear similar in highly expressed proteins (color key values > 4). (B) Average of the coefficient of variation normalized to loading for all the proteins. (C) Average and standard deviations of intensity based absolute quantification (iBAQ) values for pre-, post-synaptic and growth cone proteins in day 56 neuronal cultures.</p

RNA and immunocytochemical analysis of hNES cell-derived neuronal cultures in direct contact.

<p>RNA was isolated at different stages of differentiation (day 5 (n = 3), day 8 (n = 2), day 18 (n = 3) and day 49 (n = 3)) altogether from Line B and C. RNA expression profiles of (A) caudal (<i>CoupTF2</i>, <i>PROX1</i>), (B) medial (<i>SATB1</i>, <i>LHX6</i>) and (C) lateral (<i>MEIS2</i>, <i>GSX2</i>) ganglionic eminence markers. (D) Expression of sub-pallium (<i>ASCL1</i>), and pre-synaptic markers (<i>VGAT</i>, <i>VGLUT1</i>) overtime during differentiation. (E) Cortical progenitors (BF1, <i>TBR2</i>) and pallium marker (<i>PAX6</i>); (F) cortical upper layers (<i>CUX1</i>, <i>BRN2</i>, <i>SATB2</i>); (G) deep cortical layers (<i>TBR1</i>, <i>CTIP2</i>) and (H) rosette and neural stem cell (<i>HES5</i>, <i>DACH1</i>, <i>PLZF</i>, <i>SOX2</i>) markers were also assessed in the RNA samples. All the primers data was normalized to the expression of housekeeping gene <i>EIF4G2</i>. Error bars represent standard mean error (SEM) per time point from n = 3 in day 5, 18, 49 and n = 2 in day 8. One-way ANOVA was performed and with significant differences (p<0.05) in some gene expressions, a post hoc test represents significant differences (in asterisks) among the time points with a p<0.05. Immunocytochemical analysis at day 49 (n = 2) for Glutamatergic lineage markers CTIP2 (I), SATB2 (J), CGE marker PROX1 (K), LGE marker MEIS2 (L) and Calbindin (M) with MAP2. (N) Only the neuronal population is human-derived as shown by immunocytochemistry for human nucleus (HN), GFAP (rat astrocytes) and MAP2. Scale bars are 25 μm.</p

Mice with megalencephalic leukoencephalopathy with cysts: a developmental angle

Objective: Megalencephalic leukoencephalopathy with cysts (MLC) is a genetic disease characterized by infantile onset white matter edema and delayed onset neurological deterioration. Loss of MLC1 function causes MLC. MLC1 is involved in ion-water homeostasis, but its exact role is unknown. We generated Mlc1-null mice for further studies. Methods: We investigated which brain cell types express MLC1, compared developmental expression in mice and men, and studied the consequences of loss of MLC1 in Mlc1-null mice. Results: Like humans, mice expressed MLC1 only in astrocytes, especially those facing fluid-brain barriers. In mice, MLC1 expression increased until 3 weeks and then stabilized. In humans, MLC1 expression was highest in the first year, decreased, and stabilized from approximately 5 years. Mlc1-null mice had early onset megalencephaly and increased brain water content. From 3 weeks, abnormal astrocytes were present with swollen processes abutting fluid-brain barriers. From 3 months, widespread white matter vacuolization with intramyelinic edema developed. Mlc1-null astrocytes showed slowed regulatory volume decrease and reduced volume-regulated anion currents, which increased upon MLC1 re-expression. Mlc1-null astrocytes showed reduced expression of adhesion molecule GlialCAM and chloride channel ClC-2, but no substantial changes in other known MLC1-interacting proteins. Interpretation: Mlc1-null mice replicate early stages of the human disease with early onset intramyelinic edema. The cellular functional defects, described for human MLC, were confirmed. The earliest change was astrocytic swelling, substantiating that in MLC the primary defect is in volume regulation by astrocytes. MLC1 expression affects expression of GlialCAM and ClC-2. Abnormal interplay between these proteins is part of the pathomechanisms of MLC

Astrocytes are central in the pathomechanisms of vanishing white matter

Vanishing white matter (VWM) is a fatal leukodystrophy that is caused by mutations in genes encoding subunits of eukaryotic translation initiation factor 2B (eIF2B). Disease onset and severity are codetermined by genotype. White matter astrocytes and oligodendrocytes are almost exclusively affected; however, the mechanisms of VWM development remain unclear. Here, we used VWM mouse models, patients' tissue, and cell cultures to investigate whether astrocytes or oligodendrocytes are the primary affected cell type. We generated 2 mouse models with mutations (Eif2b5Arg191His/Arg191His and Eif2b4Arg484Trp/Arg484Trp) that cause severe VWM in humans and then crossed these strains to develop mice with various mutation combinations. Phenotypic severity was highly variable and dependent on genotype, reproducing the clinical spectrum of human VWM. In all mutant strains, impaired maturation of white matter astrocytes preceded onset and paralleled disease severity and progression. Bergmann glia and retinal Müller cells, nonforebrain astrocytes that have not been associated with VWM, were also affected, and involvement of these cells was confirmed in VWM patients. In coculture, VWM astrocytes secreted factors that inhibited oligodendrocyte maturation, whereas WT astrocytes allowed normal maturation of VWM oligodendrocytes. These studies demonstrate that astrocytes are central in VWM pathomechanisms and constitute potential therapeutic targets. Importantly, astrocytes should also be considered in the pathophysiology of other white matter disorders