Isolation and expansion of oligodendrocyte progenitor cells from cryopreserved human umbilical cord blood

Abstract

Background aims: Oligodendrocyte precursor cells (OPC) hold promise as a cellular therapy for demyelinating diseases. The feasibility of using OPC-based therapies in humans depends upon a reliable, readily available source. We have previously described the isolation, expansion and characterization of oligodendrocyte-like cells from fresh human umbilical cord blood (UCB). We now describe the isolation and expansion of OPC from thawed, cryopreserved UCB.

Methods: We thawed cryopreserved UCB units employing a standard clinical protocol, then isolated and plated mononuclear cells under previously established culture conditions. All OPC cultures were trypsinized at 21 days, counted, then characterized by flow cytometry after fixation, permeablization and labeling with the following antibodies: anti-oligodendrocyte marker 4 (O4), anti-oligodendrocyte marker 1 (O1) and anti-myelin basic protein (MBP). OPC were also placed in co-culture with shiverer mouse neuronal cells then stained in situ for beta tubulin III (BT3) and MBP as a functional assay of myelination.

Results: The average OPC yield per cryopreserved UCB unit was 64% of that seen with fresh UCB. On flow cytometric analysis, 74% of thawed UCB units yielded cells with an O4-expression level of at least 20% of total events, compared with 95% of fresh UCB units. We observed myelination of shiverer neurons in our functional assay, which could be used as a potency assay for release of OPC cells in phase I human clinical trials.

Conclusions: Our results demonstrate that OPC can be derived reliably from thawed, cryopreserved UCB units, and support the feasibility of using these cells in human clinical trials.

Figures

Figure 1

Morphology of cryopreserved UCB-derived OPC. In culture, cryopreserved UCB-derived OPC initially displayed bipolar morphology, then developed branching processes radiating from the cell body over the subsequent 2–3 weeks in culture.

Figure 2

Percentage of RBC in mononuclear cell suspension versus OPC yield. We found an inverse correlation between RBC percentage and OPC yield from both fresh and thawed cryopreserved UCB units. Yields from thawed, cryopreserved UCB increased almost 6-fold (6.0 × 10e6 versus 1.1 × 10e6) when units with a low RBC percentage were compared with those with high RBC percentages. R2 = 0.386.

Figure 3

Representative OPC phenotype by flow cytometry. (A, B) Co-expression of the oligodendrocyte markers O1 and MBP in (A) oligodendrocytes derived from fresh UCB and (B) oligodendrocytes derived from thawed, cryopreserved UCB. The O1 + MBP + population can be seen in the right upper quadrant. (C, D) O4 expression in (C) oligodendrocytes derived from fresh UCB and (D) oligodendrocytes derived from thawed, cryopreserved UCB. Both demonstrate positive staining.

Figure 4

In vitro functional assay of myelination by cryopreserved UCB-derived OPC. Shiverer mouse neurons co-cultured in vitro with UCB-derived OPC were co-stained for BT3 (Texas Red) and MBP (FITC). A shiverer neuronal control had positive staining for BT3 (A1) but not MBP (A2). Shiverer neuronal cells co-cultured with cryopreserved UCB-derived OPC expressed both BT3 (B1) and MBP (B2) after 1 week in co-culture. A z-stacked projection of shiverer neuronal cells co-cultured with cryopreserved UCB-derived OPC after 3 weeks in co-culture demonstrates BT3 expression (C1) as well as close association between BT3-expressing neuronal cells and MBP-expressing cells (C2). A z-stacked projection of shiverer neuronal cells co-cultured with cryopreserved UCB-derived OPC after 3 weeks in co-culture demonstrates BT3 expression (D1) as well as close association between BT3-expressing neuronal cells and MBP-expressing cells, and with MBP expression along axonal processes (C2). Images obtained using a Leica SP5 confocal microscope.