Pediatric Blood and Marrow Transplant Program, Duke University Medical Center, Durham, North Carolina 27710, USA.
Cytotherapy. 2011 Jul;13(6):722-9. doi: 10.3109/14653249.2011.553592. Epub 2011 Feb 22.
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.
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.
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.
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.
少突胶质前体细胞（OPC）有望成为脱髓鞘疾病的细胞治疗方法。基于 OPC 的治疗方法在人类中的可行性取决于是否有可靠的、易于获得的来源。我们之前描述了从新鲜人脐血（UCB）中分离、扩增和鉴定少突胶质样细胞。现在我们描述了从解冻、冷冻保存的 UCB 中分离和扩增 OPC。
我们采用标准的临床方案解冻冷冻保存的 UCB 单位，然后在先前建立的培养条件下分离和接种单核细胞。所有 OPC 培养物在第 21 天进行胰蛋白酶消化，计数，然后通过固定、通透和用以下抗体标记进行流式细胞术分析：少突胶质细胞标志物 4（O4）、少突胶质细胞标志物 1（O1）和髓鞘碱性蛋白（MBP）。还将 OPC 与震颤小鼠神经元细胞共培养，然后进行原位染色，检测β微管蛋白 III（BT3）和 MBP，作为髓鞘形成的功能测定。
每个冷冻保存的 UCB 单位的 OPC 产量平均为新鲜 UCB 的 64%。在流式细胞术分析中，与新鲜 UCB 的 95%相比，74%的解冻 UCB 单位产生的细胞具有至少 20%的总事件 O4 表达水平。我们在功能测定中观察到震颤神经元的髓鞘形成，这可作为 I 期人类临床试验中 OPC 细胞释放的效力测定。
我们的结果表明，OPC 可以从解冻、冷冻保存的 UCB 单位中可靠地获得，并支持在人类临床试验中使用这些细胞的可行性。
