Chondrogenical Differentiation of Umbilical Cord Lining. Stem Cells

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1 Chondrogenical Differentiation of Umbilical Cord Lining Stem Cells He Xi, Masilamani Jeyakumar, Phan Toan Thang Bioengineering, National University of Bioengineering Abstract Cartilage defects, such as osteoarthritis, have been playing a major role joint disease. Since body could not regenerate cartilage on its own and there is no effective surgical substitute for cartilage replacement, large amount of experiments had been conducted on cartilage generation using stem cells. In this experiment, two kinds of human cord lining stem cells were cultured with chondrogenic inducing factors for nine weeks. The morphological changed of induced Human Cord Lining Epithelial Cells (CLEC) and Human Cord Lining Mesenchymal Cells (CLMC) were observed and compared with the control group. CLECs exhibited greater morphological changed and better cell proliferation comparing with CLMCs.The morphological changes were more apparent for CLEC cells than CLMC cells. After three weeks of culturing, the cells were harvested and Safrain O staining was performed for both groups of cells. Both groups of cells did not exhibit positive result for chondrogenic protein. Introduction Stem cells are undifferentiated cells from the embryo, fetus or adult that are able to generate various differentiated tissues or cells under appropriate biochemical, hormonal and mechanical stimuli (Caplan 1991; Caplan and Bruder 2001). The embryonic stem cells are derived from embryos and are pluripotent and are able to differentiate into various types of cells and tissues. The adult stem cells are undifferentiated cells found among differentiated cell in a tissue or organ and they are multipotent. For instance, the mesenchymal stem cells (MSCs) are found in adult bone marrow and would be able to generate multiple mesoderm tissue types (Caplan 1991). The human umbilical cord stem cells are derived from human umbilical cord lining or cord blood. They are found in outer amniotic lining of the umbilical cord. The Mesenchymal stem cells (CLMC) are derived Wharton s Jelly and the Epithelial stem cells (CLEC) are derived from cord epithelium. Unlike embryo stem cells, the umbilical cord stem cells would not bring any ethical issues and they are easy to obtain in great quantity. The other advantage of human umbilical cord stem cells would be they are easy to obtain in great quantity and would be easily cultured and harvested comparing with adult stem cells. For cartilage induction, numerous studies has been done using human or animal mesenchymal stem cells with positive results (U.Matis et al. 2007; Matsumi et al. 2005) Although adult mesenchymal stem cells have the capability o differentiate into various mesenchymal tissues, they have a limited expansion and differentiation capacity (Varghese et al. 2006). New researches have

2 shown that embryonic mesenchymal stem cells have the ability to differentiate into various mesenchymal tissues (Barberi et al. 2005). The differentiation into cartilage cells would be induced by TGF-ß1. Material and methods Cell preparation and culture methods Mesenchymal stem cells (CLMC) and Epithelia stem cells (CLEC) from human donors were collected and both cultured in a 24-well plate (4 wells per role), in which 3 roles with membrane for cell sectioning. Every 3 roles of well there would be one control group and 2 roles of induction groups. During the whole experiment, the cells were kept at 37 in a humidified atmosphere containing 5% CO 2. The medium was changed at a 3-day interval and the chondrogenic inducing medium was introduced to both CLEC cells and CLMC cells on the 3 rd day. Both CLMC cells and CLEC cells cultured on membrane were harvested on the 4 th week and stored for sectioning. The rest cells were harvested on the 9 th week and Safarnin O staining was conducted to test the presence of the chondrogenic protein. Media The growth medium used was DMEM F12, into which other supplements were added in to make it Chondrogenic inducing. The basal differentialtion medium was DMEM F12 supplemented with 20ng/ml transferring Growing Factor b3, 100 nm Dexamelthasome, 50 μg/ml ascorbic acid, 100 μg/ml sodium pyruvate, 40 μg/ml Proline and ITS-Plus at the final concentrations. The ITS-Plus, a collaborative biomedical product, was composed of 6.25 mg/ml bovine insulin, 6.25 mg/ml transferin, 6.25 mg/ml selenious acid, 5.33 mg/ml linoleic acid and 1.25 mg/ml bovine serum albumin. Pellet Cultivation Pellet cell culture initiated by transferring 0.5 ml of cell solution into each well and 0.5 ml of growth medium was added into each well. 3 role of the cells were cultured with on membrane. Total two pellets were prepared, one for CLEC cell and the other for CLMC cells. The pellets were incubated for 9 weeks at 37 in 5% CO2, during which cell medium was changed every 3 days. The cells cultured on the membrane were harvested at the end of the 4 th week and stored in the fridge for later use in sectioning. Morphology Monitor Both CLEC and CLMC cells was monitored using electronic microscope every 3 days after changing the medium. Two pictures would be taken from control group and 4 from the induction group. Staining

3 Safrainin-O and Type II Collagen Stains for in vitro chonfrogenesis were used in the staining process. The CLEC and CLMC cells pellet was fixed in 10% neutral buffered formalin for 24 hours at room temperature and dehydrated with 70%, 80% and 95% ethanol. The pellets were stained with Eosin and rinsed by 100% ethanol. After the preparation, both control and induction groups were deparaffinized in xylene, 100% ethanol, 95% ethanol and 70% ethanol. Then they were stained with Weigert s iron hematoxylin and destained in fresh acid alcohol and rinsed in tap water. After that, aqueous fast green FCF staining was performed followed by aqueous Safranin-O staining. Results and Discussion Effect of growth factors on morphological changes for CLEC cells Exposure to chondrogenic inducing medium has caused great morphological change in CLECs. The control group cells tend to have a long thin bundle shape and while the induced group cells tend to have irregular shapes, for instance star shape. The percentage of irregular shaped cells was high in the induction group comparing with CLMCs, which indicate CLECs were more responsive to the induction medium and have a higher rate of chondrogenic gene expression. Another conclusion could also be drawn that the induction medium had an negative effect on cell proliferation. The cell density per unit area for induction group was visibly smaller than the control group. Fig. 1. Morphology of Induction Cells and Control Cells at Day 0

4 Fig. 2 Morphological difference between Induction Cells and Control Cells at Week 1 Fig. 3 Morphological difference between Induction Cells and Control Cells at Week 3 Fig.4 Morphological Difference between Induction Cells and Control Cells at Week 6 Effect of growth factors on morphological changes for CLMC cells The morphological change for CLECs was not as apparent as CLECs, both the control group and the induction group have a long slim shape. The shape of the induction group was flatter and irregular comparing with the control group. The percentage of irregular shaped cells was not high for CLECs, this indicated a lower rate of chondrogenic gene expression induced by the induction medium used in the experiment. The cell proliferation for CLMCs was not as good as CLECs. This would be due to the fact that CLMCs was not as responsive as CLECs to the medium.

5 Fig. 5 Morphological difference between Induction Cells and Control Cells at Week 1 Fig. 6 Morphological difference between Induction Cells and Control Cells at Week 3 Fig. 7 Morphological difference between Induction Cells and Control Cells at Week 3 Staining Result for CLECls and CLMCs The staining showed negative result for both CLECs and CLMCs. Only the blue stain and the brown stain stayed in both the induction group of cells and the control group of cell. The blue-green color stain was indicative for cytoplasm and the brown color stain was able to stain nucleus only while a reddish orange color would stay in cartilage, mucin, and mast cell granules. There was no indication of red color after staining performed on two batches of CLECs and CLMCs, which implied that although there were apparent morphological changes in both CLECs and CLMCs, the cells did not differentiated into cartilage cells. One possible reason causing this experiment result could be that the staining was not properly conducted and the red color stain was not able to stain the existing cartilage protein. To clarify this, further sectioning of the induction groups should be performed.

6 Fig 8. Staining Result for Induction Cells and Control Cells at Week 6 Fig 8. Staining Result for Induction Cells and Control Cells at Week 6 Reference 1. Chikayoshi Matsuda, Mutsumi Takagi, Takako Hattori, Shigeyuki Wakitani and Toshiomi Yoshida, Differentiation of human bone marrow mecenchymal stem cells to chondrocytes for construction of three-dimensional cartilage tissue, Cytotechnology, 2005, 47: Shyni Varghese, Paranduangji Therungsirikul, Angela Ferran, Nathaniel Hwang, Adam Canver, Jennifer Elisseeff; Chondrogenic differentiation of human embryonic germ cell derived cells in hydrogels, 2006, EMBS Annual International Conference New York City, USA, Aug 30-Sept Chang H. Lee, Eduardo K. Moioli, and Jeremy J. Mao; Fibroblastic Differentiation of Human Mesenchymal Stem Cells using Connective Tissue Growth Factor, 2006, EMBS Annual International Conference New York City, USA, Aug 30-Sept A. I. Caplan, Mesenchymal stem cells: cell-based reconstructive therapy in orthopedics, Tissue Eng., vol. 11, pp , Yonf-Soo Choi, Sang-Min Lim, Hyun-Chong Shin, Chang-Woo Lee, Sang-Lin Kim, Dong-II Kim, Chondrogenesis of human periosteum-derived progenitor cells in atelocollagen, Biotechnol Lett (2007) 29: