In vitro differentiation of umbilical cord blood CD34+ cells into mature megakaryocytes and generation of plateletss

ABSTRACT

BACKGROUND:

There still was not any report about inducing stem cells into matured cells to form products in vitro.

To induce CD34+ cells of umbilical cord blood to differentiate into mature megakaryocytes, and to investigate the mechanism of production of platelets.DESIGN, TIME AND

This cytology in vitro study was conducted at the Central Laboratory of Xiangya Hospital and Xiangya Third Hospital from 2004 to 2006. MATERIALS Umbilical cord was collected from healthy full-term pregnant puerperants at the Xiangya Hospital.

The CD34+ cells were isolated from umbilical cord blood by magnetic activated cell sorting (MACS) and then cultured in 24-well culture plate at 5x107/L in StemPro-34 serum-free medium, supplemented with L-glutamine, saturated human transferrin, CaCl2, insulin, deionized bovine serum albumin and recombinant human thrombopoietin at 37℃, under 0.05 volume fraction CO2 saturated humidity to be differentiated into megakaryocytes for 14-21 days. Cell medium was absorbed, and centrifuged to obtain supernatant. Samples were centrifuged again, and then supernatant was removed. The remaining was platelet-like particles in cell culture plate. Platelet was isolated from normal platelet-rich plasma.MAIN OUTCOME

The following parameters were measured morphological changes in cultured cells and platelet-like particles in supematant; results of immunohistochemistry; observation results under a microscope; platelet aggregation; CD41 expression.

At day 10, silk-like substances were found in megakaryocyte culture medium, with the presence of platelet-sized particles. The production of platelet-sized particles reached a peal at day 16. Cultured cells were strongly positively for platelet-specific antigen GP Ⅱb Ⅲa. Under the optical microscope, mature megakaryocytes were detected, with the presence of some immature megakaryocytes, and platelet-sized particles were found surrounding megakaryocytes. Under the electron microscope, a majority of mature megakaryocytes and a few apoptotic megakaryocytes were detected, and platelet-sized particles in the supernatant had the same size and structure with the platelet in the platelet-rich plasma. Some platelet surfaces were smooth or irregular. Platelet-sized particles in the supematant aggregated in response to thrombin as platelets in normal platelet-rich plasma. Flow cytometry demonstrated that the cultured platelets had the same high expression rate of CD41 as the platelets from platelet rich plasma.

Umbilical cord blood CD34+ cells can be induced to differentiate into pudfied and mature megakaryocytes and platelets in vitro.