Increasing evidence suggests that adult and postnatal stem cells are valuable resources for various medical therapies. Among many tissue-originated multipotent stem cells, UCMS cells are very usable due to their abundance, low immunogenicity[26
], and simplicity of harvest and in vitro
]. However, the tissue distribution characteristics of transplanted UCMS cells in the normal body have not been studied previously. Many of the aforementioned therapies require systemic transplantation of stem cells. Therefore, their tissue distribution in physiological and pathological conditions is important. Here we report the distribution of hUCMS cells in SCID mice after systemic transplantation using the sensitive tracking method of radiolabeled cell detection combined with histochemical and immunohistochemical detection of hUCMS cells.
The lung and spleen were found to be the major distribution tissues at 24 h after transplantion (Figure , white bars). The spleen continued to be the highest distribution site even 3 d after transplantation (Figure , light grey bars). Since the spleen is the site for clearance of most foreign and damaged cells present in circulation, the splenic distribution may be associated with clearing of damaged or dead hUCMS cells. Splenic clearance may also be related to the initial clearance of radioactivity in the urine after transplantation (Figure ).
On day three, in addition to the spleen, the gastrointestinal tract, including the stomach and small and large intestines, were the primary distribution organs (Figure , white grey bars). However, this distribution pattern was changed on day 7, when the highest distribution was found to be in the adrenal glands and uterus (Figure , dark grey bars). The lung, spleen and intestine show lesser amounts by day 7. This pattern was not changed at 14 d after transplantation (Figure , black bars) although the total radioactivity recovered was significantly decreased.
On the other hand, although a significant amount of radioactivity was distributed in skeletal muscle, the India Black ink labeled UCMS cells were not detected in skeletal muscle. This is perhaps due to the fact that the UCMS cells distrubuted through such a large amount of skeletal muscle. Therefore, although the total amount of calculated radioactivity in muscle is high, the specific activity (radioactivity per gram tissue) is low, and individual cells may have escaped detection. To study the distribution of hUCMS cells in bone marrow, radioactivity in whole femur homogenate was determined. A noticeable amount of radioactivity was detected in the femur homogenate. This may suggest that hUCMS cells localize in the bone marrow niche and potentially contribute to bone marrow function.
The primary distribution sites were visually confirmed by detection of hUCMS cells using immunohistochemical methods and India Black ink labeling of the hUCMS cells. Careful observation of the morphology of the transplanted cells in H&E tissue sections, immunohistochemical and India Black ink labeling and the time course study suggest that the majority of transplanted cells detected in various tissues are intact and were not engulfed by macrophages or other phagocytic cells. Transplanted UCMS cells remained as characteristically large cells and many of them were adjacent to the lung vasculature even 14 d after transplantation (Figure ). Therefore, a significant portion of the transplanted UCMS cells appears to be intact for a lengthy period. However, a portion of the transplanted cells appears to be destroyed and removed from circulation, and free tritiated thymidine is excreted into the urine. Therefore, it is difficult to rule out the possibility that a small portion of radiolabeled hUCMS cells may have been engulfed by macrophages and redistributed with macrophage migration. However, all three detection methods were in good agreement and identified the lung, gastrointestinal tract, and brain as primary tissue distribution sites at days 1 and 3 after cell transplantation. This agreement of UCMSC distribution between the three methods supports the suggestion that India Black ink labeled cells behaved as unlabeled cells do in vivo
, although it is difficult to completely exclude the possibility that India Black ink labeled cells may have been sequestered differently. The high level of radioactivity distribution to the adrenal glands may be misleading, since we were unable to detect the intact hUCMS cells in adrenal glands by immunohistochemical and histochemical methods. Since the adrenal glands and ovaries are small (approximately 30 mg/gland), a small artifact in the radioactive signal would be exaggerated mathematically. In addition, it is possible that metabolized or free tritiated thymidine may have been redistributed to these tissues. We expected to find that the lung was the primary distribution site in the early stages of transplantation because our previous studies indicated that systemically transplanted IFN-β-expressing hUCMS cells significantly reduce the growth of lung metastasized MDA 231 human breast carcinoma xenografts[19
]. This efficient lung distribution indicates that UCMS cells may have substantial therapeutic potential in various lung diseases including cancer, asthma and pneumonia, when UCMS cells are used to deliver therapeutic molecules and/or express therapeutic genes.
The distribution of hUCMS cells in the uterus and ovaries is of interest. The present study clearly indicates that the major distribution site in the reproductive organs is the uterus rather than the ovaries. Since both organs are sites of extensive remodeling during the estrous cycle[27
], the stem cells may be responding to chemoattractants similar to those associated with wound healing or tumors. The observation of relatively high distribution in the intestinal tract also correlates with localization to regions of rapid tissue turnover, since the epithelium is replaced every 4-7 d. Radiolabeled hUCMS cell distribution to uterus, ovaries, and digestive tract suggests that transplanted UCMS cells may play a role in tissue remodeling associated with aggressive cell division. Although the hUCMS cells have been shown to traffic to tumor tissues in a manner similar to bone marrow mesenchymal stem cells and neural stem cells[25
], the present results suggest that a portion of systemically transplanted UCMS cells are likely to distribute to other tissues that contain actively dividing cells.
One of the most interesting findings is that there was radioactivity in the brain 1, 3, and 7 d after transplantation. This finding was confirmed by immunohistochemical detection and India Black ink labeling of hUCMS cells. Since the cells were not detected in the blood-brain barrier vasculature, they evidently crossed the blood brain barrier. Although this barrier is comprised primarily of tight junctions between endothelial cells and poses a formidable obstacle to the delivery of many therapeutic agents to the central nervous system[28
], UCMS cells could cross this barrier and potentially be used as delivery vehicles for therapeutics to the brain. In support of this speculation, systemically administered neuronal stem cells engineered to express a pro-drug processing enzyme were found to migrate into the brain and to exhibit therapeutic properties[22
]. Furthermore, bone marrow MSC or fetal stem cells physiologically cross blood-brain barrier and play an important role in blood vessel remodeling[29
] and in healing of brain injury[30
Since detection of the radiolabeled cells is very sensitive as compared to histochemical and immunohistochemical detection of the cells, it was possible to monitor the distribution of the hUCMS cells in most tissues via radiolabeling. Immunofluorescence and India Black ink labeling made it possible to clarify the exact location of the hUCMS cells within many organs, but were less sensitive than radiolabeling. A possible explanation for this sensitivity difference is that in the radioactive method we have homogenized whole tissues whereas the immunofluorescence and India ink labeling methods were carried out on sections only 5-10 μM thick, so it is possible that cells may have been present in higher numbers in other parts of the organ.
In conclusion, intravenously administered hUCMS cells exhibited time-specific tissue distribution in SCID mice. The lung was a major distribution site at 24 h after transplantation. With time, major distribution sites changed to the gastrointestinal tract at 3 and 7 d after transplantation. In addition to the gastrointestinal tract, both ovaries and uterus were found to be distribution sites. The tissue distributions were confirmed by immunohistochemical and histological observations of hUCMS cells in tissue sections. Morphological analysis revealed that systemically administered hUCMS cells can be efficently distributed in brain. These findings indicate that UCMS cells are potentially useful tools for the treatment of various diseases in the lung, gastrointestinal tract, and brain.