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Transplantation of thrombopoietin (TPO)‐expanded cord blood CD34+ cells accelerates human platelet recovery in NOD/SCID mice. It is unknown which subpopulations of the TPO‐expanded cells mediate accelerated platelet recovery and bone marrow (BM) engraftment. In this study, the contribution of these subpopulations to human platelet appearance in the blood and BM engraftment was studied in NOD/SCID mice. Following transplantation of CD34−/CD61−/lineage− cells (Lin−), human platelets were detected in the blood of recipient mice from day 4. Both time to platelet recovery and blood platelet counts at 6 weeks after transplantation showed Lin− dose dependence. The Lin− population was virtually negative for lineage marker expression and lacked CD42b expression but was heterogeneous with regard to CD36 and CD38 expression, reflecting a population in transit but not fully committed toward the megakaryocyte (MK) lineage. Although no definitive phenotype could be established of the cells generating prompt platelet production and cells generating platelets 6 weeks after transplantation, this relatively heterogeneous Lin− population is prerequisite to accelerate platelet recovery in vivo. The interval to platelet recovery after transplantation of the CD34+ cells remaining after expansion (rCD34+) was similar to mice transplanted with nonexpanded CD34+ cells, although the total platelet counts and the engraftment levels in the BM were lower. Cobblestone area‐forming cell colony‐forming cells resided mostly in the rCD34+ population. The pro‐MK CD61+ cells did not contribute to human platelet recovery or engraftment in the BM. Our study shows that not all expanded cells appear critical for transplantation. These data support that functional characterization of the expanded cell populations is warranted to make future expansion protocols suitable for clinical application. STEM CELLS 2012;30:988–996

The choice for a biomaterial is partly based on the outcome of (cyto)toxicity assays. The rationales behind the selection of certain parameters, such as cell lines, controls, and animals, were evaluated using a positive and a negative control, and one experimental sample designed to induce intermediate toxicity. Extraction and direct contact assays were performed using human epidermal keratinocytes and mouse fibroblasts and mouse epithelial cells. Cell survival was measured with the tetrazolium salt (MTT) reduction assay. In addition, local implantation studies were performed in mice and rats. The positive control induced a high degree of toxicity in all in vitro tests performed, indicating that the toxicity observed in the direct contact assay was due to in situ extraction of toxic components. In the direct contact assay the negative control tested on the mouse fibroblasts resulted in a significant reduction of cell survival. No decrease in cell survival was found using the experimental sample. Subcutaneous implantation studies in mice showed that the positive control material induced a severe degeneration in mice. However, in rats just minimal alterations were noted. The experimental material induced moderate responses only in mice. Our results indicate that the direct contact assay provides limited additional information on the cytotoxicity of materials if certain limitations are not taken into account. For the in vivo implantation assay mice were superior to rats in detecting local toxic responses.

A single-platform flow cytometry method can repeatedly measure low numbers of circulating hPLTs in the PB of the same mouse. This method may be helpful in search of new protocols aiming at accelerating PLT recovery after CB transplantation, but also in a number of clinical settings, such as monitoring PLT reconstitution after hematopoietic stem cell transplantation.