Establishing A Proliferative Neural Cell Line By Nuclear Transfer Technique

Objectve: To construct nuclear transfer cells from karyoplasts of cortical neurons of fetal rat using cytoplasts of bone marrow-derived mesenchymal stem cells as recipient in order to establish a proliferative neural cell line as new donor cells for cell transplantation in brain.Methods: (1) Bone marrow-derived mesenchymal stem cells(MSCs) were cultured and identified with CD44, CD90, CD71 and CD11b by flow cytometer. The expression of CD133 was observed by immunocytochemical method. (2) Separation of cytoplasts of bone marrow-derived MSCs was completed by centrifugation through a Ficoll density gradient. The cytoplasts were identified by HE dyeing, Giemsa dyeing, immunofluorescence double labeling method and transmission electron microscope(TEM). The cell viability of cytoplasts was estimated by trypan blue exclusion test. (3) The neurons from the cerebral cortex of embryonic 17d rats was isolated and cultured. The cells were identificated after immunostaining with MAP2 and NeuN. (4) Separation of karyoplasts of cortical neurons was completed by centrifugation through a Ficoll density gradient. The karyoplasts were identified by HE dyeing, immunofluorescence and transmission electron microscope(TEM). The cell viability of karyoplasts was estimated by trypan blue exclusion test. (5) The nuclear transfer cells were constructed by fusing the karyoplasts of neurons with the cytoplasts of bone marrow-derived MSCs by polyethylene glycol (PEG). (6) The nuclear transfer cells were identified by immunofluorescence double labeling with Hoechst33342 and anti-CD71 antibody. (7) The proliferative potential of the nuclear transfer cells was examined by immunofluorescence double labeling with anti-BrdU antibody and Hoechst33342. The growth curve of the nuclear was examined by MTT colorimetric method. (8) The nuclear transfer cells were examined by transmission electron microscope (TEM) and scanning electron microscope (SEM). (9) CD44, CD90, CD71 and CD11b of the nuclear transfer cells was examined by flow cytometer. (10) The expression of neuronal nuclear antigen (NeuN) was examined by immunofluorescence and immunocytochemical method. (11) The expression of CD133, microtubule-associated protein 2 (MAP2) and nestin were examined by immunocytochemical method.Results: (1) Bone marrow-derived MSCs had a narrow spindle morphology and packed as whirlpool. MSCs were positive for CD44, CD90 and CD71. MSCs were negative for CD1lb and CD133. (2) It was exhibited that a great deal of cytoplasts were collected from the 15% and 18% regions after centrifuging MSCs through a Ficoll gradient by HE staining and Giemsa staining. Cytoplasts had polygonal shape or round morphology. The size of cytoplast was similar to integrated MSC. MSCs were successfully enucleated and cytoplasts were collected from the 15% and 18% regions, as determined by immunofluorescence double labeling with Hoechst33342 and anti-CD71 antibody. The ratio of viable cytoplasts estimated by trypan blue exclusion test was 99.5%. The round cytoplast was observed in 12.5-18% Ficoll gradient sample with TEM. There were endocytoplasmic reticulum (ER)and chondriosomes in it. (3) The cortex neurons of embryonic rat had polygonal morphology with several processes. MAP2 immunoreactivity was observed in cytoplasm and processes of neurons. NeuN immunoreactivity was observed in nucleus, some cytoplasm around the nucleus and proximate processes. The ratio of MAP2-postive and NeuN-postive neurons were about 90% at 6h, 3d, 6d and did not change significantly at different time. (4) It was exhibited that a great deal of karyoplasts were collected from the 22%, 25% and 30% regions after centrifuging MSCs through a Ficoll gradient by HE staining and immunofluorescence labeling with Hoechst33342. The ratio of viable karyoplasts estimated by trypan blue exclusion test was 99.6%. The round karyoplast surrounded by a very thin layer of cytoplasm enclosed within a plasma membrane was observed in 22-30% Ficoll gradient sample with TEM. (5) The nuclear transfer cells were constructed by fusing the karyoplasts of neurons with the cytoplasts of bone marrow-derived MSCs by PEG. The nuclear transfer cells have fusiform shape or polygonal morphology. A few of cells have two or more processes. Some nuclear transfer cells formed cell sphere. (6)The immunofluorescence double labeling with anti-CD71 antibody and Hoechst33342 reactivity was observed in the nuclear transfer cells, which suggested that the nucleus came from neurons and the cytoplasts came from MSCs. The efficiency of nuclear transfer was 64.8%. (7) The immunofluorescence double labeling with anti-BrdU antibody and Hoechst33342 reactivity was observed in the nucleus of the nuclear transfer cells. The passage number and time of cell culture had significant effect on MTT value. The MTT values of the second and third passage of nuclear transfer cells were higher than that of the first passage (p<0.05). The MTT value of the nuclear transfer cells on fourth day was higher than that on first day (p<0.05). (8) The fusion of neuronal nucleus and cytoplasts of MSCs was observed by scanning electron microscope (SEM). It was observed that there were many reductus on the karyolemma of nuclear transfer cell and many magnanimous endocytoplasmic reticulum by TEM. (9) The 1st to 4th passage nuclear transfer cells were positive for CD44, CD90 and CD71 and negative for CD1lb. The 1st passage nuclear transfer cells were negative for CD44. The ratio of CD44-postive cells increased with the passage. The ratio of CD44-postive nuclear transfer cells of 4th passage was 98.5%. (10) NeuN immunoreactivity was observed in cytoplasm of the majority nuclear transfer cells and some nucleus. The expression of NeuN increased significantly in 2rd passage(p<0.05), then decreased gradually with passage. The difference of the expression of NeuN between 2rd and 4th passage was significant(p<0.05). (11) Nestin immunoreactivity was observed on cytomembrane of some nuclear transfer cells. The level of the expression of nestin did not change significantly with passage, but the ratio of nestin-positve cells increased with passage. CD133 immunoreactivity was observed in cytoplasm of nuclear transfer cells. The expression of CD133 did not change significantly with passage (p>0.05). MAP2 immunoreactivity was observed in cytoplasm of nuclear transfer cells and the expression of MAP2 decreased with passage. The nuclear transfer cells of 4th passage were negative for MAP2.Conclusion: (1) Separation of cytoplasts of bone marrow-derived MSCs can be completed by centrifugation through a Ficoll density gradient. (2) Separation of karyoplasts of cortical neurons can be completed by centrifugation through a Ficoll density gradient. (3) The nuclear transfer cells can be constructed by fusing the karyoplasts of neurons with the cytoplasts of bone marrow-derived MSCs by PEG. (4) The nuclear transfer cells made by this method have proliferative potential and are positve for MAP2 and NeuN, which suggests that the nuclear transfer cells are proliferative neurons. (5) The neural stem cells (NSC) markers, such as CD133, nestin, CD44 and CD90, were observed in the nuclear transfer cells. The expression of NeuN and MAP2 decreased with passage. The ratio of nestin-positve cells and CD44-postive cells increased with passage. The nuclear transfer cells can dedifferentiated into NSC.