One of the most extensively studied populations of multipotent adult stem cells are mesenchymal stem cells (MSCs). MSCs derived from the human umbilical cord vein (HUC-MSCs) are morphologically and immunophenotypically similar to MSCs isolated from bone marrow. HUC-MSCs are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. Since neural tissue has limited intrinsic capacity of repair after injury, the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesenchymal-like stem cells from the human umbilical cord vein, and studied transdifferentiation-promoting conditions in neural cells. Dopaminergic neuronal differentiation of HUC-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulfoxide (DMSO) and butylated hydroxyanisole (BHA) in N2 medium and N2 supplement. The immunoreactive cells for -tubulin III, a neuron-specific marker, GFAP, an astrocyte marker, or Gal-C, an oligodendrocyte marker, were found. HUC-MSCs treated with bFGF, SHH and FGF8 were differentiated into dopaminergic neurons that were immunopositive for tyrosine hydroxylase (TH) antibody. HUC-MSCs treated with DMSO and BHA rapidly showed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including NeuroD1, -tubulin III, GFAP and nestin was markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after neural differentiation, we confirmed the differentiation of dopaminergic neurons by TH/-tubulin III positive cells. In conclusion, HUC-MSCs can be differentiated into dopaminergic neurons and these findings suggest that HUC-MSCs are alternative cell source of therapeutic treatment for neurodegenerative diseases.

Adult stem cells can make identical copies of themselves for long periods of time. They also give rise to many differentiated mature cell types that have characteristic morphology and specialized function. Human adult stem cells are the attractive raw materials for the cell/tissue therapy, however, it is not easy to get from the adult tissues. In the present study, we tried to isolate a cell population derived from human umbilical cord vein which has been discarded after birth. The cells were isolated after treatment of the umbilical vein with collagenase or trypsin. After 3 days of culture, two kinds of cell populations were found consisting of adherent cells with endothelial cell-like and fibroblast-like morphology, respectively. When these cells were subcultured 12 times over a period of 3 months, almost cells appeared uniformly to exhibit fibroblastoid morphology which was different from that of mesenchymal stem cells obtained from human bone marrow The results of RT-PCR analyses showed distinct expression of BMP-4, oct-4, and SCF genes but not of GATA, PAX-6 and Brachyury genes. On immunohistochemical staining, the cells were negative for the von Willebrand factor(vWF), alpha-smooth muscle actin and placental alkaline phosphatase. From these observations, it is suggested that stem-like cells might be present in human umbilical cord vein.

Coculture of HSC with bone marrow-derived mesenchymal stem cells (BM-MSCs) is one of used methods to increase cell numbers before transplant to the patients. However, because of difficulties to purify HSCs after coculture with BM-MSCs, it needs to develop a method to overcome the problem. In the present study, we have examined whether a culture insert placed over a feeder layer might support the expansion of HSCs within the insert. cells isolated from the umbilical cord blood by using midiMACS were divided into three groups. A group of 1 cells were grown on a culture insert without feeder layer (Direct). The same number of HSCs was directly cocultured with BM-MSCs (Contact). The third group was placed onto an insert below which BM-MSCs were grown (Insert). To distinguish feeder cells from HSCs, BM-MSCs was pre-labeled fluorescently with PKH26 and 1 cells were seeded in the culture dishes. After culture for 13 days, the expansion factor (x) of HSCs that were grown without feeder layer (Direct) was In contrast, the number of HSCs directly cocultured with feeder layer was 59.6 0.5 and that of HSCs cultured onto an insert was The percentage of BM-MSCs cells remained being fluorescent was after culture. Immune-phenotypically large proportion of cultured cells were founded to be differentiated into myeloid/monocyte progenitor cells. The ability of BM-MSCs, fetal lung, cartilage and brain tissue cells to support ex vivo expansion of HSCs was also examined using the insert. After 11 days of coculture with each of these cells, the expansion factor of HSCs was 15.0, 39.0, 32.0 and 24.0, respectively. Based upon these observations, it is concluded that the coculture method using insert is very effective to support ex vivo expansion of HSCs and to eliminate the contamination of other cells used to coculture wth HSCs.