Glial progenitor cell-based repair of the dysmyelinated brain: Progression to the clinic

Abstract

Demyelinating disorders of the central white matter are among the most prevalent and disabling conditions in neurology. Since myelin-producing oligodendrocytes comprise the principal cell type deficient or lost in these conditions, their replacement by new cells generated from transplanted bipotential oligodendrocyte-astrocyte progenitor cells has emerged as a therapeutic strategy for a variety of primary dysmyelinating diseases. In this review, we summarize the research and clinical considerations supporting current efforts to bring this treatment approach to patients.

Keywords: Cell transplant; Cuprizone; Demyelinating disease; Glial progenitor; Leukodystrophy; Multiple sclerosis; Neural stem cell; Oligodendrocytic progenitor.

Figure 1. Perinatal hGPC grafts myelinate the congenitally unmyelinated shiverer brain

A. A 1-year-old shiverer mouse, transplanted at birth with 3 × 10⁵ human glial progenitor cells, stained for myelin basic protein (MBP, green). B. higher power view of myelinated human oligodendrocytes in the corpus callosum of a 12 week-old transplanted shiverer (MBP, green, human nuclei, red). C. By 9 months, essentially all transcallosal axons have myelinated (mouse axons in red, stained for neurofilament; human MBP, green). D. Human GPC-derived oligodendrocytes normalized nodal architecture at nodes of Ranvier, here in the cervical spinal cord of an adult, neonatally-engrafted shiverer (Caspr2, green; Contactin, blue; ßIV-spectrin, red). E, hGPC-derived oligodendrocytes produced ultrastructurally normal myelin; corpus callosum, 12 wks. Scales: A, 1 mm; B, 50 μm; C, 10 μm; D, 20 μm; E, 1 μm. Adapted from (Windrem et al., 2008).

Figure 2. Human glial progenitor cells colonize and then dominate human glial-chimeric mouse brains

A. 9-month old mouse engrafted neonatally with human GPCs, shows predominance of human GPCs over time. Human-specific NG2, green; mouse NG2, red. B. Higher power of A, showing dense engraftment of human GPCs. C. Cortical strips show progressive dominance of human GPCs (green) relative to mouse (red) at 3 (left), 9 (center) and 13 (right) months after neonatal engraftment. Scales: A, 400 μm; B, 100 μm; C, 150 μm. Adapted from (Windrem et al., 2014).

Figure 3. Human GPCs differentiate as myelinogenic oligodendrocytes in response to cuprizone demyelination

A, Mice were transplanted with 2×10⁵ hGPCs perinatally, and at 17 wks of age placed on either a cuprizone (CZN)-supplemented or normal diet for 12 wks, then either sacrificed or returned to standard diet and killed at later time-points. B–C. Serial coronal sections comparing dot-mapped distributions of human (human nuclear antigen, hN) cells in control (B) and cuprizone-fed mice at 49 wks of age, after 20 weeks recovery on control diet. D–E. Relative abundance of human (red dots) and mouse (blue) transferrin (TF)-defined oligodendroglia, in 20 μm coronal sections of corpus callosa of mice engrafted with hGPCs neonatally, demyelinated as adults from 17–29 wks of age, then assessed at 49 wks, 20 wks after cuprizone. E shows an untreated control, age-matched to D. F. The density of human cells in the corpus callosum increases to a greater degree in cuprizone-demyelinated brains than in untreated controls, including during the period of cuprizone treatment, indicating progenitor mobilization. G, By 8 weeks after the termination of cuprizone exposure, the density of human oligodendroglia is >5-fold greater in cuprizone-demyelinated than untreated control brains. H, By that 8-week recovery point, most hGPCs engrafted in the corpus callosa of cuprizone-treated mice differentiated as oligodendrocytes, and accordingly (I), over half of all transferrin-defined oligodendrocytes were human; in contrast, relatively few human oligodendrocytes were noted in untreated chimeric brains. J, Substantial colonization by human glia is evident in this remyelinated callosum, after a 20-week recovery (human nuclear antigen, magenta; myelin basic protein, green). K, chimeric white matter populated, post- cuprizone, by human GPC-derived oligodendroglia. Anti-human nuclear antigen (hNA) (red), transferrin, (green); inset shows relative abundance of hNA⁺/transferrin⁺ human oligodendroglia. Scale: J, 100 μm; K, 50 μm, inset, 25 μm. From (Windrem et al., 2020).

Figure 4. Single cell RNA-seq analysis of human glia derived from hGPC-engrafted mice

Shiverer mice were engrafted neonatally with FACS-isolated, hESC (H9)-derived CD140a⁺ hGPCs. Mice were killed at 19 weeks, and cells isolated via FACS from the dissected corpus callosum (n=3), then captured on a Chromium Controller (10X Genomics) followed by single cell 3’ (Chromium v2) library construction, and deep sequenced on an Illumina HiSeq 4000. A. Cell population clustering via t-Distributed Stochastic Neighbor Embedding (t-SNE) identified major cell types and subpopulations thereof (APC: astrocyte progenitor cells), allowing downstream differential expression analysis. B. Violin plot of lineage marker expression. Besides the canonical transcription factor markers of stage-specific glial phenotype, the strong linkage of cyclin D1 (CCND1) to the GPC stage is noted. C Pseudotime analysis ordered cells from the progenitor state to maturity, and predicts the branch point at which oligodendrocytic vs. astrocytic fate is determined at the hGPC stage. Plot shows differential expression as a function of time-discriminated fate-specific genes. From Mariani, Schanz and Goldman, 2020, unpublished data.

Figure 5. RNA-seq identifies cuprizone treatment-induced differential gene expression in hGPCs and oligodendroglia

Bulk RNA-sequencing was done on hGPCs sorted via FACS from the corpus callosa of human glial chimeras. The mice were engrafted with fetal tissue-derived hGPCs at birth, given oral cuprizone (CZN) or a control diet for 12 wks beginning at 12 wks of age, and killed at 36 wks for expression profiling. A subset of differentially-expressed genes is shown, with comparison between the hGPC, immature OL, and mature OL pools identified in the human glial scRNA-seq data of Fig 4. After gene ontology network analysis, major differentially-expressed genes were segmented into functional modules (M1–4). M1: myelination, TCF7L2 signaling; M2: Notch and TGFβ signaling, cell movement; M3: lipid and T3 transport, RXRA signaling; M4: iron and copper homeostasis, calcium signaling. Expression values are experiment-specific gene Z-Scores (red, high expression; green, low expression). From (Windrem et al., 2020).

Figure 6. Manufacturing hGPCs from hESCs

The first part of the GMP manufacturing process is focuses on initiation from the hESC master cell bank (MCB) on Day -20, followed by hESC expansion, EB formation, and neural induction with neurosphere formation by day 26, and neural stem cell expansion through day 36. Glial induction starts on Day 36, with in-process testing at days 100, 135, and 160, and hGPC recovery with cryopreservation at 160 days. Methods as described (Wang et al., 2013), as adapted for GMP compliance. From Chandler-Militello et al., unpublished.