Modeling vanishing white matter disease with patient-derived induced pluripotent stem cells reveals astrocytic dysfunction

Abstract

Aims: Vanishing white matter disease (VWM) is an inherited leukoencephalopathy in children attributed to mutations in EIF2B1-5, encoding five subunits of eukaryotic translation initiation factor 2B (eIF2B). Although the defects are in the housekeeping genes, glial cells are selectively involved in VWM. Several studies have suggested that astrocytes are central in the pathogenesis of VWM. However, the exact pathomechanism remains unknown, and no model for VWM induced pluripotent stem cells (iPSCs) has been established.

Methods: Fibroblasts from two VWM children were reprogrammed into iPSCs by using a virus-free nonintegrating episomal vector system. Control and VWM iPSCs were sequentially differentiated into neural stem cells (NSCs) and then into neural cells, including neurons, oligodendrocytes (OLs), and astrocytes.

Results: Vanishing white matter disease iPSC-derived NSCs can normally differentiate into neurons, oligodendrocytes precursor cells (OPCs), and oligodendrocytes in vitro. By contrast, VWM astrocytes were dysmorphic and characterized by shorter processes. Moreover, δ-GFAP and αB-Crystalline were significantly increased in addition to increased early and total apoptosis.

Conclusion: The results provided further evidence supporting the central role of astrocytic dysfunction. The establishment of VWM-specific iPSC models provides a platform for exploring the pathogenesis of VWM and future drug screening.

Keywords: astrocytes; induced pluripotent stem cells; neural stem cells; neurons; oligodendrocytes; vanishing white matter disease.

Figure 1

Features of the two VWM patients. a, Brain MRI of patients VWM1 and VWM2 and an age‐matched control, performed at the age of 4, 3, and 4, respectively: A, E, and I (T1WI); B, D, F, K, J, and L (T2WI); C, G, and K (T2 FLAIR). Brain MRI of both VWM patients showed symmetric abnormal signals on T1WI, T2WI, and T2 FLAIR in the white matter (WM), which was partially rarefied. b, Direct sequencing analysis of the genomic DNA from the two VWM patients’ iPSCs showed VWM1: EIF2B5 c.1827_1838del (p. Ser610_Asp613del), c.1157G>A (p.Gly386Val); VWM2: EIF2B3 c.140G>A (p. Gly47Glu), c.1037T>C (p. Ile346Thr)

Figure 2

Characterization of VWM iPSCs. a, Positive alkaline phosphatase staining showed typical morphology of iPSC clones (top) and immunochemical analysis of pluripotent markers, SSEA4, and NANOG (bottom). b, Representative hematoxylin and eosin staining of teratomas derived from the established VWM iPSC clones. The teratomas were formed via the subcutaneous injection of undifferentiated iPSCs into the posterior leg of NOD/SCID mice. VWM1: Open arrow, cartilage; asterisks, respiratory epithelia; arrow, muscle. VWM2: Arrowhead, adipocyte; asterisks, gut‐like epithelia; open arrowhead, pigmented epithelia. The scale bar represents 200 μm. c, Karyotype analysis showed normal karyotypes of VWM iPSCs (more than 10 passages), 46, XY and 46, XX, respectively

Figure 3

Differentiation of iPSC‐derived NSCs into neurons. a, Immunochemical analysis of iPSC‐derived NSCs, NSCs were positive for Nestin and SOX2. b, Mean fluorescence densities of Nestin of NSCs, no significant difference exists, P > 0.05. c, Representative image of the immunochemistry of the NSC‐derived neurons. The neurons were positive for βIII‐tubulin (green color) and Neurofilament H (red color) after 7 d of NSC differentiation. The scale bar represents 30 μm. d, Mean fluorescence densities of βIII‐tubulin and Neurofilament H of neurons; no significant difference exists, P > 0.05. e, Quantification of the early and total apoptosis of the neurons showed no significant differences between the Control, VWM1, and VWM2 neurons (P > 0.05, biological replicates, n = 3)

Figure 4

Differentiation of iPSC‐derived NSCs into oligodendrocytes. a, Schematic presentation of the protocol for OLs differentiation from NSCs. b, Immunochemical analysis of iPSC‐derived OPCs and OLs. Both control and VWM OPCs were positive for NG2 (green color) and PDGFRα (red color) after 6 d of NSCs differentiation. And both control and VWM OLs were positive for MBP after 4 d of OPCs differentiation. The scale bar represents 30 μm. c, d, Mean fluorescence densities of NG2 and MBP respectively; no significant difference exists, P > 0.05. e, Apoptosis detection of OLs via Annexin V/PI staining. Quantification of the early and total apoptosis of the OLs showed no significant differences between the controls, VWM1, and VWM2 OLs (P > 0.05, biological replicates, n = 3)

Figure 5

Differentiation of iPSC‐derived NSCs into astrocytes. a, Schematic presentation of the protocol for astrocytes differentiation from NSCs. b, Cells were positive for Nestin and CD44 after 8 d of NSC differentiation into astrocytes. The scale bar represents 30 μm. c, Mean fluorescence densities of Nestin and CD44, respectively; no significant difference exists, P > 0.05. d, Both control and VWM Astrocytes were positive for GFAP (green color) and S100β (red color) after 16 d of NSCs differentiation into astrocytes. The scale bar represents 30 μm. e, Mean fluorescence densities of GFAP and S100β, respectively; no significant difference exists, P > 0.05

Figure 6

Involvement of VWM iPSC‐derived astrocytes. a, Representative image of the immunochemistry of control and VWM astrocytes. The mature astrocytes were positive for GFAP after 28 d of NSCs differentiation into astrocytes. The scale bar represents 30 μm. b, Calculated length of the longest astrocytic process and number of processes of the astrocytes (10 fields were randomly selected, with at least 8 cells in each field). c, Immunochemical analysis of the expression of αB‐Crystalline in control and VWM astrocytes. The scale bar represents 30 μm. d, Calculated positivity of αB‐Crystalline‐positive (αB‐Crystalline/Hoechst) astrocytes (**, P < 0.0001, 10 fields were randomly picked, with at least 8 cells in each field). e, Real‐time quantitative PCR analysis for GFAP (total, αGFAP and δ‐GFAP) expression in Control and VWM astrocytes (**, P < 0.01 in the three groups, biological replicates, n = 3). f, The total GFAP expression in Control and VWM astrocytes detected by Western blot exhibited no significant difference. g, Apoptosis detection in the astrocytes via Annexin V/PI staining and quantification of total and early apoptosis of astrocytes (** represents P < 0.01, * represents P < 0.05, biological replicates, n = 3)