CD140a identifies a population of highly myelinogenic, migration-competent and efficiently engrafting human oligodendrocyte progenitor cells

Abstract

Experimental animals with myelin disorders can be treated by transplanting oligodendrocyte progenitor cells (OPCs) into the affected brain or spinal cord. OPCs have been isolated by their expression of gangliosides recognized by mAb A2B5, but this marker also identifies lineage-restricted astrocytes and immature neurons. To establish a more efficient means of isolating myelinogenic OPCs, we sorted fetal human forebrain cells for CD140a, an epitope of platelet derived growth factor receptor (PDGFR)α, which is differentially expressed by OPCs. CD140a(+) cells were isolated as mitotic bipotential progenitors that initially expressed neither mature neuronal nor astrocytic phenotypic markers, yet could be instructed to either oligodendrocyte or astrocyte fate in vitro. Transplanted CD140a(+) cells were highly migratory and robustly myelinated the hypomyelinated shiverer mouse brain more rapidly and efficiently than did A2B5(+)cells. Microarray analysis of CD140a(+) cells revealed overexpression of the oligodendroglial marker CD9, suggesting that CD9(+)/CD140a(+) cells may constitute an even more highly enriched population of myelinogenic progenitor cells.

Figure 1. CD140a identifies a proliferating oligodendrocyte progenitor in fetal cortex and intermediate zone

CD140a/PDGFRα⁺ cells were found in the cortical mantle of 22 wk gestational age (g.a.) human fetal brain. A, All identifiable CD140a⁺ cell bodies co-expressed the oligodendrocyte lineage transcription factor Olig2. B, Consistent with an early neural progenitor phenotype, CD140a⁺/PDGFRα⁺ cells within the intermediate zone co-expressed the progenitor-expressed transcription factor sox2. C, A proportion of PDGFRα⁺/CD140a⁺ cells co- labeled with Ki67 antibody, a marker of cells in active cell cycle. The main panel shows a confocal z-stack (CD140a, red; Ki67, green); the inset shows fluorophore-specific single optical sections of the imaged cell. D, shows a low power schematic reconstruction of the distribution of CD140a/PDGFRα-immunoreactive cells in a section of 18 week forebrain, showing the broad dispersal of these cells through the intermediate zone and cortical mantle. Scale: A–C, 20 μm; D, 1 mm.

Figure 2. CD140a/PDGFRα recognizes a population of cells in the fetal brain that accumulate with gestational age in the intermediate zone and cortex

Flow cytometry was used to determine the relative abundance of PDGFRα⁺ cells in the fetal human germinal zones and overlying intermediate zone and cortex. A, B, shows a typical CD140a cytometry of a 21 wk fetal cortical/IZ dissociate. A, shows limited non-specific staining using a PE-conjugated isotype control; B, the same dissociate stained using PE-conjugated anti-CD140a. CD140a⁺ cells were uncommon in the second trimester intermediate zone and cortex before 16 weeks g.a.; their incidence increased thereafter with gestational age (n=29) (C). In contrast, the incidence of CD140a⁺ cells remained relatively constant throughout the second trimester in the VZ/SVZ (n=10) (D). E shows a superimposition of all CD140a incidence data, as multiple regression lines including whole forebrain as well as dissected VZ/SVZ and IZ/cortex.

Figure 3. CD140a/A2B5/PSA-NCAM cytometry

Fetal dissociates were immunomagnetically selected on the basis of PSA-NCAM antigenicity. PSA-NCAM⁻ cells were then subject to two-color FACS for CD140a and A2B5 using PE- and APC-conjugated antibodies, respectively (A–C). Positive selection gates were defined using fluorescence-minus one (FMO) controls by substitution of either A2B5 (A) or CD140a (B) antibodies with a matched isotype control conjugated antibody. A2B5 and C140a-specific antibodies were combined in C. A large proportion of CD140a⁺ cells co-expressed A2B5 (C). PSA-NCAM⁺ cell underwent single color CD140a cytometry/FACS (not shown). D, shows the proportion of CD140a⁺ cells in each A2B5/PSA-NCAM sorted fractions (n=4, 19–22wk gestational age). The A2B5⁺PSA-NCAM⁻ fraction contained significantly more CD140a⁺ cells than unsorted dissociate (1-way ANOVA followed by Dunnett’s multiple comparison test, p<0.05). E, each sorted fraction was cultured in T3/0.5% PD-FBS media for 7 days and assessed for the immature oligodendrocyte antigen O4. CD140a⁺ cells from each A2B5/PSA-NCAM fraction gave rise to a higher proportion of O4⁺ oligodendrocytes than matched CD140a⁻cells (n=3 samples).

Figure 4. CD140a-sorted cells mature primarily as oligodendrocytes but can be maintained as bipotential progenitors

Sorted CD140a⁺ cells were plated onto substrate and allowed to differentiate following removal of exogenous growth factors. A, by 4 days after FACS, these cells had developed characteristic immature oligodendrocyte morphology expressing the sulfatide antigen O4. The proportion of O4⁺ oligodendrocytes was determined at 4 days in vitro (B). Virtually no O4-defined oligodendrocytes were found in CD140a-depleted cultures; in contrast, approximately 40% of CD140a⁺ cells developed O4 expression by that point. C, 7 days post-sort some oligodendrocytes had matured further with elaborate oligodendrocyte profiles stained with O1 antibody. D, CD140a-sorted cells were cultured for 7 days in the presence of PDGF-AA and FGF-2 (20 ng/ml each), then immunophenotyped. Under these conditions, CD140a⁺ cells continued to express A2B5, while less than 10% developed O4⁺ expression. In contrast, the CD140a⁻ fraction was comprised primarily of βIII-tubulin⁺ neurons. Scale = 10 μm.

Figure 5. CD140a sorted cells can efficiently myelinate shiverer axons

Human fetal CD140a^+/PDGFRα⁺ OPCs were transplanted into the hypomyelinated forebrain of neonatal, rag2 null-immunodeficient shiverer mice. At 8 weeks post injection, a fraction of human cells recognized by anti-human nuclear antigen (hNA, red) differentiated into myelinating oligodendrocytes expressing the myelin protein gene MBP (green) (A). Consistent with the time course of human myelination, a large proportion of transplanted CD140a⁺ cells remained as NG2-expressing OPCs at 8 weeks post-implantation (B), while a minor fraction differentiated as astrocytes, as immunolabeled by GFAP (C). D illustrates a quantification of human CD140a⁺ cell fate, at 8 weeks post implantation (n=3). Scale = 20 μm.

Figure 6. CD140a-sorted OPCs myelinate more effectively than A2B5-sorted OPCs

A, Computer-assisted drawings of 14 μm sections taken at 0.67 mm intervals throughout the forebrain of a 12 week old shiverer × rag2-null mouse, transplanted bilaterally in the corpus callosum with 100,000 cells sorted on CD140a/PDGFRα. Each red dot represents an individual cell labeled with anti-human nuclear antigen. B, The corpus callosum of an engrafted shiverer mouse at 12 weeks, stained for myelin basic protein (MBP, green), showing substantial donor-derived myelin. C, A photomicrograph of the corpus callosum and fimbria in another engrafted mouse; MBP, green. D, An individual oligodendrocyte, stained for anti-human nuclear antigen (red) and MBP (green). E–F, Ensheathment of host mouse axons (neurofilament, green; MBP, red) at 12 weeks by human fetal cells sorted on the basis of either CD140a/PDGFRα(E) or A2B5-immunoreactivity (F), manifesting the more rapid and robust axonal myelination by CD140a⁺ cells than by A2B5-sorted cells. G–H, Sections of a CD140a-engrafted shiverer callosum at 12 weeks, immunostained for MBP (red), human GFAP (green), and human nuclear antigen (blue in G, yellow in H). These images show the robust production of hGFAP⁺ astrocytes as well as MBP⁺ oligodendrocytes from the engrafted CD140a⁺ cells in vivo. Scale: B, 500 μm; C, 200 μm; D, 10 μm; E–F, 20 μm.