Construction of human cell culture system for regenerative medicine without using animal components

脐血造血干细胞的数量不足以满足高体重患者的需要，因此在不分化的情况下扩增脐血造血干细胞是非常重要的。此外，在人胚胎干细胞的情况下，没有鼠成纤维细胞就无法培养。因此，我们尝试开发不使用动物饲养细胞的人干细胞培养系统。1.与癌症研究中心合作，建立了新的人骨髓细胞与端粒酶基因整合。克隆了一些细胞系，这些克隆支持脐带血中造血干细胞（HSC）的生长。然而，在这些情况下，细胞系的生长太快而不能从细胞系中分离HSC。2.部分支持HSC生长的细胞经诱导分化为脂肪细胞后，其支持HSC生长的能力消失。因此，研究了分化前后蛋白质的差异，并通过双向电泳观察到一些差异。现在，基因表达也通过DNA微阵列研究。当蛋白质或基因用于支持HSC生长时，将蛋白质固定在人工细胞培养基质的基底上。3.将猴胚胎干细胞（ES细胞）培养在未分化的人胎盘饲养层上。考虑到胎盘通常被作为医疗废物丢弃，这一成功是非常有用的。虽然猴ES细胞只是作为临床前研究，但其相似性可以推广到人ES细胞，在再生医学的实际应用中将是非常有用的。4.将小鼠ES细胞在各种人工基质上培养，并对其生长情况进行评价。结果表明，化学固定的饲养细胞支持小鼠ES细胞的生长。目前将这种固定方法应用于猴胎盘ES细胞的培养。

It is very important to expand the number of human hematopoietie stem cells in cord blood without differentiation, because the cell number is not enough for patients of heavy weight. In addition, in the case human embryonic stem cell there is no way to culture without murine fibroblast cells. Therefore, we have tried to develop culture systems of human stem cells without using animal feeder cells.1.New human bone marrow cells incorporated with telomerase gene were established with cooperation with Cancer Research Center. Some cell lines were cloned and the clones supported growth of hematopoietie stem cells(HSC) in cord blood. However, in these cases, the growth of cell line was too fast to isolate HSC from the cell line. Therefore, the cell line was chemically fixed and employed for supporting growth of HSC.2.When some cells supporting growth of HSC were induced to fat cells, the supporting abilities disappeared. Therefore, the difference of proteins before and after the differentiation was investigated and some differences were observed by two dimensional electrophoresis. Now the gene expression is also investigated by a DNA microarray. When the protein or gene for supporting growth ofHSC, the protein will be immobilized on a substrate for artificial cell culture matrix.3.Monkey embryonic stem(ES) cell was cultured on human placenta feeder layers without differentiation of ES cells. Considering that he placenta was usually discarded as a medical waste, the success is very useful. Although the monkey ES cell was used as a preclinical research, the similarity can be extended to human ES cells and it will be very useful for practical regenerative medicine.4.Mouse ES cells were cultured on various artificial substrata and the growth was evaluated. It was found that chemically fixed feeder cell supported the growth of murine ES cells. Now this fixation is applied for placenta for culture of monkey ES cells.