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

扩增脐带血中的人造血干细胞数量非常重要，因为对于体重较大的患者来说，细胞数量是不够的。此外，在人类胚胎干细胞的情况下，没有小鼠成纤维细胞是无法培养的。因此，我们尝试在不使用动物饲养层细胞的情况下建立人类干细胞的培养体系。1.与肿瘤研究中心合作建立了新的携带端粒酶基因的人骨髓细胞。克隆了一些细胞系，克隆的细胞支持脐带血中的造血干细胞(HSC)生长。然而，在这些情况下，细胞系生长太快，无法将HSC从细胞系中分离出来。当一些支持HSC生长的细胞被诱导为脂肪细胞时，支持HSC的能力消失。因此，我们研究了分化前后蛋白质的差异，并用双向电泳法观察到了一些差异。现在，基因表达也通过DNA微阵列进行了研究。当获得支持HSC生长的蛋白质或基因时，将该蛋白质固定在人工细胞培养基质上。3.将猕猴胚胎干细胞培养在人胎盘饲养层上，未分化为ES细胞。考虑到胎盘通常被当作医疗废物丢弃，这一成功是非常有用的。虽然猴ES细胞被用作临床前研究，但其相似性可以扩展到人类ES细胞，这将对实用的再生医学非常有用。4.将小鼠ES细胞在不同的人工基质上进行培养，并对其生长进行评估。研究发现，化学固定的饲养层细胞对小鼠ES细胞的生长有支持作用。现在，这种固定方法被应用于培养猴子ES细胞的胎盘。

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.