Isolation,culture and identification of rat bone marrow mesenchymal stem cells and endothelial progenitor cells

Yuhui YANG; Jixiong WU

ABSTRACT

BACKGROUND:

There are no unified methods to isolate,culture and amplify bone marrow mesenchymal stem cells(BMSCs) . Endothelial progenitor cells(EPCs) can be harvested by inducing peripheral blood or bone marrow mononuclear cells. However,the method of collecting bone marrow mononuclear cells is various,and the isolation efficiency is different.

OBJECTIVE:

To isolate and identify BMSCs and EPCs in vitro. DESIGN,TIME AND

SETTING:

The cytological in vitro study was performed at the Central Laboratory,First Affiliated Hospital,Anhui Medical University from October 2008 to February 2009. MATERIALSTwo clean healthy male Sprague Dawley rats were supplied by the Anhui Animal Center.

METHODS:

Rat bone marrow was extracted to harvest mononuclear cells by density gradient centrifugation. BMSCs were isolated by the differential adhesion. Following 48 hours of primary culture of BMSCs,non-adhered cells were collected and induced by EGM-2 complete medium supplemented with fetal bovine serum,vascular endothelial growth factor,fibroblast growth factor,insulin-like growth factor-1 and recombinant human epidermal growth factor. There were three groupsearly adherence + common culture EPCs,twice adherence + induction culture EPCs,and rat mature aorta EPCs. Nitric oxide content was directly measured in the medium using nitrate reductase method. MAIN OUTCOME

MEASURES:

The following parameters were measuredin vitro culture of BMSCs and EPCs,and nitric oxide content in the medium.

RESULTS:

Primary culture of BMSCs was in a spindle-shape,within 24 hours the majority of cells were adherent,9-10 days up to 90% cells were confluent. Purified BMSCs were amplified and uniform,long spindle,with the passage cycle for 8 days. Flow cytometry detection showed that cells were CD34-,CD45-negative and CD105-positive. Second adherent of EPCs induced by 3 days culture showed colony formation following six days,appeared cord-like structure and microvessel-like growth at 8-10 days. At 2 weeks,the majority of cells were polygonal,colony-forming unit was interconnected,showing a typical "cobblestone"-like. At 7-10 days,cells were double stained DiI-acLDL and FITC-UEA-1. Flow cytometry showed Flk-1-positive and CD133-positive. Nitric oxide content was significantly higher in the culture medium compared with the ordinary cultured cells(P