All-trans retinoic acid-induced nerve cell differentiation of rat bone marrow mesenchymal stem cells inhibits T lymphocyte proliferation

Hanning ZHAO; Fang LIU

ABSTRACT

BACKGROUND:

Several studies have demonstrated that bone marrow mesenchymal stem cells (BMSCs) inhibit the proliferation and activation of T lymphocytes, exerting a role of negative immunological regulation.

OBJECTIVE:

To induce rat BMSCs to differentiate into neuron-like cells in vitro using all-trans retinoic acid and to investigate the effects of differentiated cells on T lymphocyte proliferation.DESIGN, TIME AND

SETTING:

A cytological in vitro observation was performed in Guangdong Medical College in March 2008.MATERIALS Two male 4-week-old Sprague Dawley rats were provided by the Laboratory Animal Center of Guangdong Medical College. Fresh healthy human blood was provided by the Department of Clinical Laboratory, Guangdong Medical College.

METHODS:

Rat BMSCs were isolated by adherent method and were passaged in vitro. Cells of passage 5 were pre-induced with Low glucose-dulbecco's modified eagle's medium (LG-DMEM) containing 0.3 mg/L all-trans retinoic acid and 10% fetal bovine serum. Twenty-four hours later, aforementioned LG-DMEM was discarded and LG-DMEM containing 0.6 mg/L all-trans retinoic acid was added to induce cells differentiation into neuron-like cells. Fresh healthy human blood was taken to prepare responding cells. Rat BMSCs, as stimulator cells, were included for one-way mixed lymphocyte reaction. Four groups were designated. Control group 100 μL of responding cells at a concentration of 1×109/L; Experimental group 11×104 neuron-like cells + 100 μL of responding cells; Experimental group 21×105 neuron-like cells + 100 μL of responding cells; Experimental group 31×106 neuron-like cells + 100 μL of responding cells.MAIN OUTCOME

MEASURES:

Morphological observation of induce-differentiated cells, identification of nerve cells, and one-way mixed lymphocyte reaction results.

RESULTS:

Fifty minutes after addition of inducing medium, under the optical microscope, BMSCs exhibited a typical morphology of perikaryon. Three hours later, most of cells became into bipolar or multipolar neuron-like cell appearance with cell bodies and processes. Immunocytochemical staining results showed that majority of cells exhibited neuron specific enolase- and nestin-positive expression and glial fibrillary acidic protein-negative expression. Compared with the control group, radionuclide counts per minute were significantly reduced in each experimental group (P<0.05). There was significant difference in radionuclide counts per minute between experimental groups (P<0.05). With increasing BMSCs amount, the inhibitory effects on T lymphocyte proliferation were more evident.

CONCLUSION:

AII-trans retinoic acid can induce neuron-like cell differentiation of rat BMSCs in vitro. The neuron-like cells can inhibit human T lymphocyte proliferation in a dose-dependent manner.