A trial for reconstitution of planarians from cultured neoblasts.

1994 Fiscal Year Final Research Report Summary

• Project/Area Number: 05808070
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: Developmental biology
• Research Institution: Himeji Institute of Technology
• Principal Investigator: AGATA Kiyokazu Himeji Institute of Technology Faculty of Science Associate Professor, 理学部, 助教授 (70167831)
• Project Period (FY): 1993 – 1994
• Keywords: Planarian / Regeneration / Neoblast / Cell culture

Research Abstract

It has been belived that planarians store a lot of the totipotent stem cells (neoblasts) in their bodies and other cells can not proliferate than neoblasts. Here, I have tried to culture the neoblasts of planarians in vitro and to reconstitute planarians from cultured neoblasts. If such a culture system is established, we could analyze the relations of the gene function to cell organization more easier.
1.Isolation of molecular markers for neoblasts
To find out the molecular markers for neoblasts, randomly selected 200 cDNA clones were analyzed by DNA sequencing. In these clones cdc5 and histone genes of planarians were identified. These genes were expected as marker genes for proliferating cells. However, in situ hybridization using these cDNAs as probes did not show any significant signal. A ATP-dependent RNA helicase gene was also identified, which has high similarity to the Drosophila vasa gene. The vasa product is known as a component of germ plasm which is indispensable for mainten ance of the totipotency of the germ cells. This gene is also expected as a marker to identify neoblasts. Now antibodies against the planarian vasa-like protein are being prepared to identify the cells expressing the vasa-like gene.
2.Cultures of planarian dissociated cells
We have succeeded in culture of planarian dissociated cells in vitro. Neurons could attach on the dishes and elongate axons. It was also observed that several macrophage-like cells were moving on the dishes. However, it was very difficult to proliferate them in culture. We could not get any evidence that dissociated cells can divide in our culture conditions. We also prepared the extracts of planarians and fractionated them into several fractions by gel filtration. Although a activity stimulating axon growth was detected in the extracts, any fraction of the extracts did not stimulate proliferation of planarian cells in vitro. When planarian cells were dissociated in the presence of Ca2+, epithelial cells could not dissociate into a single cell. they could be isolated as single layred sheets and then formed spherical bodies.