| Besides human embryonic stem cells, there are some pluripotent adult stem cells in human body, which can differentiate into tissue cells to replace the old or dead cells and maintain the structure and the function of the normal tissue. For example, Mesenchymal Stem cells (MSC), when cultured in reasonable conditions, can differentiate into osteoblast, cartilage cells, adipose cell, muscle cells or nerve cells, etc. It was reported that there are some adult stem cells in umbilical cord blood such as CD34+ hemopoietic stem cells, MSC and CD34+ cells-derived endothelial cell precursors. Success in transplantation therapy of malignancy hematopathy and immunodeficiency disease using CD34 hemopoietic stem cells (HLA mismatch or partial match) suggests a promising application of the umbilical cord blood clinically. Because of the convenient source of umbilical cord blood, no harm to patients or neonates and no essential problem of ethics and morality, umbilical cord blood cells are widely used in science research and clinical study. Only CD34+ hemopoietic stem cells are generally accepted as adult stem cells up to now. Whether there exists some new kind of stem cell in umbilical cord blood has not been reported.According to the present reports, the amount of stem cell is very low in umbilical cord blood. However, it is very difficult to isolate and purify the stem cells in umbilical cord blood from mononuclear cells using routine methods. Methods of efficient culture and induction of new stem cells are critical to the application of stem cells in umbilical cord blood. Based on three fundamental hypotheses (1.The routine stem cells isolation method is not fit for all kinds of stemcells. 2. Hyperosmotic culture may be a means of culture and screening stem cells. 3. Different cells and different developmental stages are maintained in different basal nutrition components and dosages.), we innovate a new culture medium of stem cells (named Potentculture medium, the patent number: 200410016898.8). A new kind of pluripotent stem cell named PSC3445 (the patent number: 200410016897.3) was established from human umbilical cord blood using the Potentculture medium. The specific culture method of umbilical cord blood PSC3445 cell is named "to cultivate first and then to attach". The following was studied:1. Mononuclear cells from human umbilical cord blood were cultured in the hyperosmotic Potentculture medium and uniform fibroblast-like cells were established. These cells can easily attach to plastic flask but difficultly to glass culture flask. Potentculture medium can selectively improve the proliferation of the PSC3445 cells, while it can inhibit growth of the monocyte cells. Potentculture medium can be the screening medium for PSC3445 cells.2. Umbilical cord PSC3445 cells had very strong proliferation ability. The cells under mitosis primarily divided along longitudinal axis or horizontal axis. 1 107 PSC3345 cells could be generated using 50-100ml umbilical cord blood in vitro. PSC3445 cells grew in a "whirlpool" pattern without cell confluence. High concentration of cytokines such as IL-6, IL-8 was detected in the supernatant of the growth media.3. Umbilical cord PSC3445 cells expressed many surface markers of monocyte such as CD11b, CD14, CD45, etc. CD34, CD90, CD166 were not detected in the surface of the cells. Electric microscopy examination showed many cilia around the cell and an obvious nucleus. Chemical staining showed that the cells are periodic acid-Schiff (PAS) positive, Oil Red O (ORO) negative, alkaline phosphatase (Alk.p) negative, and Peroxidase (POX) negative. Most of the PSC3445 cells wereacid phosphatase (AP) positive.4. We have induced these cells to differentiate into mature macrophages by LPS (lipopolysaccharide, the specific inducing agent of monocytes and macrophages), T lymphocytes by IL-2 (Interleukin-2, the specific inducing agent of T lymphocytes), epithelial cells by EGF (epidermal growth factor, the specific inducing agent of epithelial cells), neuronal cells by NG...
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