Isolation and Expansion of Oligodendrocytes from Thawed, Cryopreserved Human Umblilical Cord Blood

Abstract

While transplantation of unrelated umbilical cord blood (UCB) halts the progression of neurological damage in children with lysosomal storage diseases (LSD), it does not necessarily reverse the damage sustained before transplant. Isolation and expansion of central nervous system progenitor cells from UCB has potential therapeutic application in treating these patients. We have recently described the reliable isolation and expansion of oligodendrocyte-like precursor cells (OPC) from freshly collected, non-crypreserved UCB (Tracy et al. Cytotherapy . 2008, 3:1–8). We anticipate utilizing these cells as adjuvant, targeted therapy to reduce the time to donor cell correction/prevention of disease-induced CNS injury. This approach will require use of cyropreserved donor UCB. We now demonstrate the feasibility of isolating and expanding OPCs from a series of thawed, cryopreserved UCB units and comparing those cells to OPCs derived from fresh UCB units. Cryopreserved UCB units were thawed using a standard protocol employed in clinical transplantation. Mononuclear cells were isolated by either ficoll density separation or centrifugation after a dextran-albumin wash. Fresh UCB units underwent hetastarch depletion of red blood cells then mononuclear cell isolation by ficoll density gradient separation. Both types of cells were plated at 3×10(6) cells/ml in media containing platelet derived growth factor, neurotopin 3, vascular endothelial growth factor, and triiodothyronine. All UCB unit cultures were trypsinized at 21 days, counted, then characterize by flow cytometry after being fixed, permeablized, and labeled with the following antibodies: anti-oligidendrocyte marker 4 (O4), anti-oligidendrocyte marker 1 (O1), anti-myelin basic protein (MBP). To examine phenotypic changes over time, cultures from two units (one thawed, one fresh) were also analyzed weekly over 4 weeks. On flow cytometric analysis, 72% of thawed UCB units yielded O4-expressing cells as at least 20% of total events compared with 94% of fresh UCB units (Table 1). Average oligodendrocyte yield per UCB unit was less for thawed units (9.65×10(5)) compared with fresh (3.77×10(6)). However, within the gated population, expression of O1, O4, and MBP was similar between thawed and fresh cords. We also noted early expression of the preoligodendrocyte marker O4 by 1–2 weeks in culture, followed by increasing expression of mature oligodendrocyte markers O1 and MBP over the subsequent 2–4 weeks (Table 2). Our results demonstrate that oligodendrocyte precursor cells can be derived reliably from thawed, cryopreserved UCB units, and suggest the feasibility of using these cells in human clinical trials.Table 1: Characteristics of UCB-Derived OligodendrocytesThawed UCB UnitsFresh UCB UnitsTotal Units Cultured1816Mean # mononuclear cells plated/UCB Unit2.55×10(8)1.4×10(8)Final oligodendrocyte cell count/UCB Unit8.41×10(5)3.7×10(6)Units with O4 expression >20% of total events13 (72%)15 (94%)Events in gate as % total events58.2677.83O4 expression (% of gated)83.6686.45O1 expression (% of gated)88.1386.29MBP expression (% of gated)31.1031.41Co-expression of MBP-O1 (% of gated)28.5031.86Table 2: Expression of Markers by UCB-Derived OligodendrocytesThawed UCB UnitFresh UCB UnitDayO1MBPMBP+ O1DayO1MBPMBP+ O1Day 795.7510.149.45Day 768.7825.1312.11Day 1498.0735.9335.74Day 1497.7134.0333.75Day 2195.2435.4331.80Day 2199.2234.8234.72Day 2899.6951.0851.04Day 2896.4924.1023.40