A comparison of haematopoietic stem cells from umbilical cord blood and peripheral blood for platelet production in a microfluidic device

Abstract

Background and objectives: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB).

Materials and methods: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively.

Results: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0·0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 ± 9% versus 76 ± 11%, respectively (P < 0·0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7·4 platelet-like particles per input cell from PB compared to 4·2 from UCB (P = 0·02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes.

Conclusion: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank.

Keywords: haematopoiesis; megakaryopoiesis; platelets; stem cells; tissue engineering.

Figure 1

Static cell culture of HSC derived from PB or UCB. (a) The percentage HSC obtained by magnetic isolation per total nucleated cells in UCB (□) or PB (●) samples. (b) The proliferation (all cells), in function of time, expressed as fold increase relative to the number of cells seeded on D0. (c) Expression of CD34 and (d) CD42b/CD41 markers on cultured cells as measured by flow cytometry as a function of time. Data are shown as mean with SD (n ≥ 5). Statistical analysis results are shown as *P < 0·05, **P < 0·01, ***P < 0·001 and ****P < 0·0001.

Figure 2

Platelet production in hydrodynamic flow. (a) Representative image of cell anchorage (*) to VWF‐coated pillars (white dots) of a cultured cell (D12) started from UCB and (b) PB. Cell elongation and (pro)platelet formation is indicated by red arrows. The flow direction is shown by a white arrow. (c) The number of PLP produced per input cell during the perfusion experiment. Data are given as mean with SD (n ≥ 12). Statistical analysis results are shown as *P < 0·05. (d and e) The PLP yield was plotted as a function of the number of CD42b/CD41 double positive input cells for (d) UCB (n = 36) and (e) PB (n = 13).

Figure 3

Expression of platelet markers. The fraction of PLP that was staining positive for (a) CD42b, (b) CD41, (c) CD42b/CD41 and (d) CD61 produced from UCB (open bars) and PB (closed bars). Data are shown as mean with SD (n ≥ 5).

Figure 4

Number of platelet receptors. The number of (a) CD42b, (b) CD61 and (c) CD49b receptors was determined using GP screen kit on PLP produced from UCB (open bars) and PB (closed bars). Data were compared to the levels in bPLT (hatched bars). Data are shown as mean with SD (n = 4).

Figure 5

Differential interference microscopy. Representative images of the cell suspensions from (a) UCB or (b) PB after perfusion and double centrifugation. PLP are indicated by arrows. The larger cells were most likely MK (*). (c) Representative image of bPLT prepared in the same way as the PLP. Pictures were taken from DIC microscopy studies 1000× magnification.