Osteoblast Progenitor Cells Derived from Umbilical Cord Blood 167 μ μ α α μ β μ μ α μ μ μ μ β μ

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1 Differentiation of Osteoblast Progenitor Cells from Human Umbilical Cord Blood Seung-Jin Hong 1, Eun-A Lee 3, Gue-Tae Chae 2 and Hoon Han 1 Departments of 1 Microbiology and Immunology, 2 Pathology, College of Medicine, The Catholic University of Korea, 3 Histostem Corporation, Seoul, Korea ABSTRACT Background: Human umbilical cord bloods, which could be taken during the delivery are utilized as a source of hematopoietic stem cells. Also in cord blood, there are several kinds of stem cells such as endothelial and mesenchymal stem cells. Methods: We isolated the mesenchymal stem cells from human umbilical cord bloods and confirmed the differentiation of these cells into osteoblast progenitor cells. The mesenchymal stem cells derived from umbilical cord blood have the ability to differentiate into specific tissue cells, which is one of characteristics of stem cells. These cells were originated from the multipolar shaped cells out of adherent cells of the umbilical cord blood mononuclear cell culture. Results: The mesenchymal stem cells expressed cell surface antigen CD13, CD90, CD102, CD105, α-smooth muscle actin and cytoplasmic antigen vimentine. Having cultrued these cells in bone formation media, we observed the formation of extracellular matrix and the expression of alkaline phosphatase and of mrna of cbfa-1, ostoecalcin and type I collagen. Conclusion: From these results we concluded that the cells isolated from the umbilical cord blood were mesenchymal stem cells, which we could differentiate into osteoblast when cultured in bone formation media. In short, it is suggested that these cells could be used as a new source of stem cells, which has the probability to alternate the embryonic stem cells. (Immune Network 2002;2(3): ) Key Words: Human umbilical cord blood, mesenchymal stem cells, osteoblast progenitor cell Immune Network 166

2 Osteoblast Progenitor Cells Derived from Umbilical Cord Blood 167 μ μ α α μ β μ μ α μ μ μ μ β μ

3 168 Seung-Jin Hong, et al. μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ α Table I. Name and nucleotide sequences of the primers Primer Sequences Tm o C Bp GAPDH 1 sense 5'-ACCACAGTCCATGCCATCAC-3' GAPDH anti-sense 5'-TCCACCACCCTGTTGCTGTA-3' 55.9 ALP 2 sense 5'-ACGTGGCTAAGAATGTCATC-3' 48.8 ALP anti-sense 5'-CTGGTAGGCGATGTCCTTA-3' 49.8 Type I collagen sense 5'-TGACGAGACCAAGAACTG-3' 45.2 Type I collagen anti-sense 5'-CCATCCAAACCACTGAAACC-3' 53.8 Cbfa-1 sense 5'-CCGCACGACAACCGCACCAT-3' 65.5 Cbfa-1 anti-sense 5'-CGCTCCGGCCCACAAATCTC-3' 63.3 Osteocalcin sense 5'-CCTGAAAGCCGATGTGGTC-3' Osteocalcin anti-sense 5'-CTCACACTCCTCGCCCTAT-3' 68 1: Glycerophosphatase dehydrogenase, 2: Alkaline phosphatase

4 Osteoblast Progenitor Cells Derived from Umbilical Cord Blood 169 A B C D E F Figure 1. Morphologies of adherent mononuclear cells obtained from human umbilical cord blood. A. After 6 days of culture, suspension and adhesion cells were found ( 100), B. After 17 days of culture, spindle shaped cells grew rapidly ( 100), C. Culture 3 wks. The spindle shaped cells grew confluently and filled up the flask ( 100), D. Culture 1 month. The spindle shaped cells grew up to form multilayer ( 100), E. Treated with bone formation media for 2 wks. ( 100), F. Treated with bone formation media for 1 month. Massive matrix formation could be seen. ( 100). α

5 170 Seung-Jin Hong, et al. Figure 2. FACS results of spindle shaped cells. The CD34, CD45, CD14 were not expressed and the CD90, CD105, CD13 were expressed. The stromal cell cytoskeleton vimentine, and alpha-smooth muscle actine were expressed. Collagen type I could not be defined by cytoplasmic staining. Control, isotype antibody treated group without bone formation media.

Osteoblast Progenitor Cells Derived from Umbilical Cord Blood 171 A B C D E F G H Figure 3. Results of immunofluorescence staining on the spindle shaped cells ( 400). A. CD90, B. CD105, C. CD102, D.

6 Osteoblast Progenitor Cells Derived from Umbilical Cord Blood 171 A B C D E F G H Figure 3. Results of immunofluorescence staining on the spindle shaped cells ( 400). A. CD90, B. CD105, C. CD102, D. α-smooth muscle actin, E. intracellular cytoskeleton vimentine, F. CD13, G. Intracellular collagen type I in bone formation media non-treatment group, H. Intracellular collagen type I in bone formation media treatment group. A B Figure 4. Expression of alkaline phosphatase in spindle shaped cells. A. bone formation media non-treated group ( 100). B. bone formation media treated group ( 100). Figure 5. The expression of mrna of alkaline phosphatase, type I collagen, cbfa-1 and osteocalcin were checked in bone formation treated and non-treated groups of passage 3 stage of multipolar shaped adherent cells.

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