Optimal condition of directional-differentiation of neurons from retinoic-acid induced MSESPU35 embryonic stem cell lines

Maolin QIN; Wenqin CAI; Zhongxiang YAO

ABSTRACT

BACKGROUND:

Neural axon regeneration is one of the difficulties that must be overcome in treatment of injury of central nerve system. Significant therapeutic effects have been obtained in transplantation of neural stem cells (NSCs), embryonic stem cells (ESCs) and Schwann cells. But the bottleneck situation of insufficiency of cell provider has limited the development on it.

OBJECTIVE:

To observe directional-differentiation of retinoic-acid induced ESCs so as to find optimal condition for neuronal differentiation.

DESIGN:

Non-randomized controlled experiment was designed.

SETTING:

Teaching-Research Room of Histology and Embryology, Department of Basic Medicines. Third Military Medical University of Chinese PLAMATERIALS The experiment was performed in Staff Room of Histology and Embryology, Third Military Medical University of Chinese PLA from January to May 2000. Eighteen Kunming mice in disoestrus were employed, of which. 12 mice were female and 6 mice male. They were placed in same cage at ratio of 21 for mating. The date of pregnancy was recorded. MESPU35 ESC line was prepared.

METHODS:

Removed head. internal organs and four limbs, feeder-layer Feeder-layer adherent culture was used to proliferate MESPU35 ESCs.Classic 4-/4+ method [The embryoid body (EB) grew naturally for 4 days,without retinoic acid added. In the coming 4 days, retinoic acid was added to induce neural EB of high proportion] was applied to induce the directional differentiation of the nerve. EB was cultured with serum of different concentrations. Phase contrast microscope was used to observe nerve-like EB in serum of different concentrations and to count numbers. ②Immunocytochemical technique was used to observe cellular morphological charac ters at various differentiating phase spots (5th. 9th, 14th days) and with retinoic acid at various concentrations. Flow cytometer (FCM) was used to count the proportion of differentiated neurons.MAIN OUTCOME

MEASURES:

①Estimated measurement of the length of process and cell body during formation of neural EB after retinoic-acid induced differentiation of MESPU35 ESCs. ② Observation of cell morphology with immunocytochemical staining and proportion of differentiated cells assayed with FCM.

RESULTS:

①It was discovered with phase contrast microscope that serum of different concentrations affect neural directional differentiation after EB formation to certain extent. Excessively high and low concentrations of serum reduced the proportion of neural differentiation of EB. The differentiating proportion is high in serum with 5% concentration. ② It is observed with immunocytochemical technique that the proportions of NF200 positive cell and glial fibrillary acidicprotein (GFAP) positive cell in differentiation of MESPU35 ESCs induced by retinoic acid were increased with phase spots in differentiation and increased concentration of retinoic acid. NF200 positive cell is transformed as multipolar neurons from absence of process in morphology. The processes of GFAP positive cell became longer and linked among each other as reticular pattern finally. ③ It was assayed with FCM that the proportion changes of GFAP positive cell and NF200 positive cell manufactured in differentiation were similar to immunocytochemical one.

CONCLUSION:

Retinoic acid in combination with proper concentration of serum and differentiating phase spots can induce neural-differentiation of MESPU35 ESC at high proportion and its differentiating regulation is in the patterns of concentration dependence and time dependence.