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

Xin, Li
Fang-ping, Chen
Jing, Liu
Xin-hua, Wu
Tie-bin, Jiang
Xue-yuan, Tang

¹Department of Hematology, Xiangya Third Hospital, Central South University, Changsha 410013, Hunan Province, China;
²Department of Hematology,
³Department of Obstetrics, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China

Correspondence to: Chen Fang-ping, Professor, Doctoral supervisor, Department of Hematology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China [email protected]

Received:2008-07-29

Accepted:2008-11-20 (54200807170019/ZS)

Li X, Chen FP, Liu J, Wu XH, Jiang TB, Tang XY.In vitro differentiation of umbilical cord blood CD34+ cells into mature megakaryocytes and generation of platelets. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2009;13(10): 1976-1980(China)

Journal of Clinical Rehabilitative Tissue Engineering Research 13(10):p 1976-1980, March 5, 2009.

Abstract

BACKGROUND:

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

OBJECTIVE:

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 SETTING:

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.

METHODS:

The CD34⁺ cells were isolated from umbilical cord blood by magnetic activated cell sorting (MACS) and then cultured in 24-well culture plate at 5×10⁷/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 °C, 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 MEASURES:

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

RESULTS:

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 supernatant 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.

CONCLUSION:

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

In vitrodifferentiation of umbilical cord blood CD34+ cells into mature megakaryocytes and generation of platelets☆