Studies for Stability of Ex Vivo Produced Megakaryocytes and Their Distribution in Transplanted Mice

We previously established an efficient pilot-scale system to generate sufficient megakaryocytes/platelets for potential clinical applications. Here, we present recent studies that demonstrate the feasibility for stable preservation of ex vivo generated megakaryocytes/platelets (MKs/PLTs) and analyze distribution of these cells in transplanted NOD/SCID mice.

The 8-day ex vivo culture of megakaryocyte progenitors (MKPs) were harvested and cryopreserved in liquid nitrogen for 1 or 3 months. Cells were then thawed and further cultured for 5 days so that these cells were differentiated toward the megakaryocytic lineage. Meanwhile, matured MKs/PLTs generated from fresh cord blood CD34⁺ cells on day 13 were collected and stored at 4°C with plasma substitution for 1, 3, 5, and 7 days, respectively. Subsequently, cell viability and specific bio-marker expression were analyzed for their phenotypic stability at various stored conditions. To observe distributions of ex vivo produced MKs/PLTs with transfected lenti-GFP in sub-lethally irradiated NOD/SCID mice (n=5) at the dose of 1.0×10⁷ transfected cells/mouse. After transplantation for 0.5, 3, 6, and 24 h, mouse organs were harvested and images were immediately taken. Furthermore, peripheral blood of each mouse was collected and human GFP⁺CD41a⁺ MKs and GFP⁺CD42b⁺ PLTs were detected by flow cytometry to evaluate platelet-releasing potential of transplanted MKs in vivo . Mice transplanted with no GFP-labelled cells were used as negative control (n=3). Mice injected with saline alone were used as blank control (n=3).

After 5 days' culture of MKPs that had been cryopreserved for 1 or 3 months, cell viability was 79.0%±3.3% or 75.2%±5.6%, with CD41a purity of 85.3%±4.5% or 87.2%±3.9% and CD61 purity of 68.4%±4.1% or 63.2%±2.3%, respectively. Cell viability and bio-marker expression of cryopreserved MKPs after in vitro differentiation were similar with 13-day culture of matured MKs/PLTs from freshly collected cord blood CD34⁺ cells (Viability: 85.5%±2.7%; CD41a: 82.4%±6.1% and CD61:74.0%±5.5%, respectively). For mature MKs/PLTs stored at 4°C, the cell viability of these four groups was 81.2%±4.5%, 74.3%±5.6%, 73.2%±4.9%, and 71.5%±6.3%, respectively; Percentages of CD41a cells of these four groups were 62.7%±5.1%, 58.3%±4.4%, 59.1%±6.0%, 56.9%±7.7% whereas percentages of CD61 cells were 65.8%±3.9%, 60.7%±2.5%, 45.2%±1.9%, 49.4%±4.4%, respectively. These results strongly suggest that MKPs of 8-day culture can be cryopreserved in liquid nitrogen for 3 months or longer, and matured MKs/PLTs of 13-day culture can be stored at 4°C for 7 days or longer without significantly affecting their viability and immunophenotypic characteristics. It is important to note that none of the groups had any bacterial and fungal contaminations before or after storage and during subsequent culturing processes. The fact that these cells were free of endotoxin and pyrogen strongly suggests that ex vivo generated MKs/PLTs are safe and stable. In mouse studies, no fluorescent signal could be detected in the blank control group, and only a trace amount of auto-fluorescence was detected in lung of the negative mouse group. However, in experimental group strong GFP signals could be observed in lung of each mouse 0.5 h after transplantation with the peak fluorescence between 3 and 6 h. Lung GFP signals remained detectable 24 h post transplantation. In mouse peripheral blood, human GFP⁺CD41a⁺ MKs could be detected only in the first 0.5 h after transplantation, and then human CD41a⁺CD42b⁺ PLTs emerged at 0.5 h with a low level of 0.5%±0.1%, after which the percentage increased rapidly with a peak level of 3.2%±0.6% at 6 h, indicating that the transplanted human MKs started to release PLTs after they merged into lung. Besides lung, a very weak signal was observed in the kidneys and livers, and completely disappeared at 24 h in mice of the experimental group. These results imply that engrafted mature MKs are capable of migrating into lung for further maturation, releasing platelets in NOD/SCID mice.

MKPs of 8-day culture can be cryopreserved in liquid nitrogen for at least 3 months without affecting their viability and phenotypic characteristics. Mature MKs/PLTs of 13-day culture are stable in plasma substitute at 4°C for 7 days. The transplanted human MKs/PLTs appear to primarily home to the lung of NOD/SCID mice to release platelets in vivo .