Mean PLT volume correlated with current markers of CB hematopoietic potential and is potentially useful for evaluating CB collections for banking. The question of the clinical significance of PLT characteristics in CB transplantation remains unanswered.

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BACKGROUND: Growth of megakaryocytic lineage in vitro from isolated CD34+ cells from fresh cord blood (CB) has been in the focus of recent research, whereas megakaryocytic potential of thawed unseparated CB bank products has remained less well characterized. The aim of this study was to evaluate further the megakaryocytic potential of unseparated thawed CB cells in liquid and collagen matrices using thrombopoietin (TPO), interleukin 3 (IL-3) and IL-6. MATERIALS & METHODS: Viability (trypan blue), nucleated cell (NC) and CD34+ cell concentration and colony-forming cells (CFC) were assessed pre-freezing and post-thawing from CB units processed and thawed by current CB bank procedures. NCs from unseparated thawed CB units (red blood cell and plasma reduced before freezing; n=5; range 0.06–0.5x106/ml; CD34+ cell range 0.2–3.5x103/ml) and fresh CB (Ficoll separated mononuclear cells, n=3; 0.1x106/ml; CD34+ cell range 0.3–0.8x103/ml) were cultured in protein free medium (PF01, MacoPharma) supplemented with albumin, insulin and transferrin at 37°C for 9–15 days and in semisolid medium (MegaCult-C, StemCell Technologies Inc.) for 14 days. Megakaryocytic colony formation was evaluated as total score (0–3, 0=no growth, 3=maximum growth; Eskola et al. Transfusion2008;48:884-892). Flow cytometry, cytocentrifugation and immunohistochemical staining were used to detect hematopoietic and megakaryocytic cells. RESULTS: Viability of NCs varied from 83 to 99% and the recovery of CD34+ cells from 76 to 147% post-thawing. Clonogenic progenitors were preserved after thawing. Total fold increase of cells was up to 27 and 10 in cultures of thawed and fresh cells, respectively. Megakaryocytic cells were observed clearly on cytospin slides at days 12–14. Notably, CD41+ cell aggregates of similar size as megakaryocytic cells were observed already at day 5. In flow cytometry, the amount of large cells with moderate and high CD41 expression increased up to 7-fold from day 5 on in liquid cultures of both thawed and fresh cells. Proportions of CD34+ cells among large CD41+ cells were 76% (mean; range 61–87%, n=3) and 62% (mean; range 35–75%, n=3) for cultures of thawed and fresh CB at days 12–15. Throughout the culture particles of the same size as adult platelets were present. Megakaryocytic growth potential of different thawed CB units was variable in liquid culture, whereas good growth from each CB unit was detectable in semisolid collagen matrix. The scores varied from 2.0–2.5 for thawed and 1.8–3.0 for fresh cells. Both thawed and fresh CB units with the largest numbers of NCs, CD34+ cells and total CFC proliferated well and produced the largest amounts of CD41+ cells. The liquid as well as semisolid culture conditions also allowed proliferation of other hematopoietic cell lines. CONCLUSIONS: Unseparated cells from thawed CB units can produce megakaryocytic cells in vitro. Megakaryocytic differentiation potential of thawed cells in liquid culture varies between the CB units. At its best it was comparable to megakaryocytic growth potential in collagen matrix when identical exogenous cytokines were applied. Current CB bank procedures seem not to cause freezing lesions detectable on the phenotype of megakaryocytic progenitors in short term cultures. Distinguishing cell aggregates from large megakaryocytes requires careful morphological analysis. Best growth of megakaryocytic cells was detectable in cultures of CB units with largest numbers of NCs and CD34+ cells.

OBJECTIVE: Delayed platelet engraftment is a common problem after hematopoietic stem cell transplantation. However, although aiming at high quality, the megakaryocytopoietic potential of cord blood (CB) units is not routinely studied in CB banks. In this study, a simple method for the evaluation of the megakaryocytic growth of unfrozen CB units was designed. The results were compared with cell characteristics of the units, including mean platelet volume (MPV) and platelet concentration in unprocessed CB as possible markers of thrombopoietic potential. MATERIALS & METHODS: Buffy coat (BC) samples from 24 randomly selected CB units were cultured on two double-chamber slides per unit in a semisolid assay designed to support megakaryocytic cells (MegaCult-C, StemCell Technologies Inc., Vancouver, Canada). After 13 days, the cells were fixed and stained using a monoclonal CD41 antibody and the alkaline-phosphatase anti-alkaline-phosphatase technology. Based on the appearance of megakaryocytic colonies, the CB units were scored on a scale 0–3 by three independent investigators. The scores were compared with CB bank data of the same units. The nonparametric Kendall coefficient of concordance (W) and simple linear regression were used for statistics. RESULTS: The study sample was confirmed representative of all CB units processed during the same time period. All CB units received a mean score of at least 0.7, indicating megakaryocytic growth. The scores given by the three investigators appeared similar and a high concordance was observed (W= 0.91; p < 0.0001), although the distribution of scores varied slightly between the investigators. Using simple linear regression, a clear yet not statistically significant correlation was discovered between the megakaryocyte (MK) score and the volume proportion of platelets in unprocessed CB (“plateletcrit”), defined as MPV × platelet concentration.A correlation (although not statistically significant) was also observed between the MK score and both MPV and platelet concentration. MK score and markers of thrombopoiesis (n=24) Mean (Range) R p* *Two sided Plateletcrit, % 0.23 (0.13–0.35) −0.21 0.32 MPV, fl 8.9 (8.0–9.7) 0.27 0.20 Platelet concentration, 109/l 258 (136–374) −0.25 0.23 In addition, a negative correlation between MPV and platelet concentration was discovered (R= −0.35; p= 0.091). No significant correlation was observed between the MK score and total colony-forming unit (CFU) number, total CD34+ cells or total nucleated cells (TNC) in BC. MK score and CB unit cell contents (n=24) Mean (Range) R p* *Two sided Total CFU, 106 2.4 (0.8–4.5) 0.004 0.98 Total CD34+ cells, 106 5.4 (1.4–11.5) 0.04 0.84 TNC, 107 124 (69.4–212) 0.13 0.54 CONCLUSIONS: As the megakaryocytopoietic potential of a CB unit does not necessarily correlate with data used to select units for banking and transplantation, screening for this potential may be of clinical significance. The screening method used in this study was simple and required no extra sample from the CB unit; however, more studies are needed to identify the best procedure for a routine setting. The associations observed between the MK score, MPV and platelet concentration may reflect the autoregulation of thrombopoiesis as well as the reliability of the screening procedure. The development of screening methods for the megakaryocytopoietic potential of a CB unit may be an important step in improving platelet engraftment after transplantation.

Immediately post-thaw, CBUs possess an ability to generate large BFU-like megakaryocytic colonies, whereas the colonies were not detectable in most CBUs in semisolid culture after a short suspension culture. Small CFU-Mks were observed throughout the cultures. It may be that the BFU-Mk colonies matured and acquired CFU-Mk behaviour.