J Korean Med Sci.  2010 Oct;25(10):1418-1426. 10.3346/jkms.2010.25.10.1418.

Stemness Evaluation of Mesenchymal Stem Cells from Placentas According to Developmental Stage: Comparison to Those from Adult Bone Marrow

• Sung HJ
• Hong SC
• Yoo JH
• Oh JH
• Shin HJ
• Choi IY
• Ahn KH
• Kim SH
• Park Y
• Kim BS

• KMID: 2157843
• DOI: http://doi.org/10.3346/jkms.2010.25.10.1418

Abstract

This study was done to evaluate the stemness of human mesenchymal stem cells (hMSCs) derived from placenta according to the development stage and to compare the results to those from adult bone marrow (BM). Based on the source of hMSCs, three groups were defined: group I included term placentas, group II included first-trimester placentas, and group III included adult BM samples. The stemness was evaluated by the proliferation capacity, immunophenotypic expression, mesoderm differentiation, expression of pluripotency markers including telomerase activity. The cumulative population doubling, indicating the proliferation capacity, was significantly higher in group II (P<0.001, 31.7+/-5.8 vs. 15.7+/-6.2 with group I, 9.2+/-4.9 with group III). The pattern of immunophenotypic expression and mesoderm differentiation into adipocytes and osteocytes were similar in all three groups. The expression of pluripotency markers including ALP, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and telomerase were strongly positive in group II, but very faint positive in the other groups. In conclusions, hMSCs from placentas have different characteristics according to their developmental stage and express mesenchymal stemness potentials similar to those from adult human BMs.

Keyword

Stemness; Placenta; Bone Marrow; Mesenchymal Stem Cells

MeSH Terms

Antigens, Surface/metabolism
Bone Marrow Cells/*cytology/metabolism
Cell Proliferation
Female
Humans
Immunophenotyping
Mesenchymal Stem Cells/*cytology/metabolism
Mesoderm/cytology
Octamer Transcription Factor-3/metabolism
Placenta/*cytology/growth & development
Pregnancy
Pregnancy Trimester, First
Proteoglycans/metabolism
Stage-Specific Embryonic Antigens/metabolism
Telomerase/metabolism

Figure

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  Fig. 1 Cells from the human placentas and human bone marrow. (A) Cells from the human term placenta. (B) Cells from the first-trimester placenta. (C) Mesenchymal stem cells(MSC) from the human bone marrow. The placenta derived cells show a fibroblastoid, spindle-shaped morphology at passage 3 on day 5. The growth pattern of the cells from the term and first-trimester placenta was same as the bone marrow derived mesenchymal stem cells. Phase contrast: magnification ×100 for all figures.

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  Fig. 2 Proliferation Capacity. (A) Cumulative population doublings of Mesenchymal stem cells from first-trimester human placenta (hPC), term human placenta (hPC) and Bone marrow. (B) The possible passages for subcultures with the maintenance of initial morpholology and growth pattern of three population of mesenchymal stem cells.

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  Fig. 3 Immunophenotype of MSCs. BM-derived MSCs (BM-MSC), first-trimester placenta derived cells and term placenta derived cells were labeled with antibodies against the indicated antigens, and analyzed by flow cytometry. Representative histograms are demonstrated. All types of cells showed the same immunophenotypic characteristics: positive expression of CD29, CD44, CD90, CD105 and HLA-ABC as surface epitopes, and negative expression of CD31, CD34, CD45 and HLA-DR.

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  Fig. 4 Osteogenic differentiation from placenta derived cells. For osteogenic differentiation, first-trimester placenta derived cells were cultured for up to 3 weeks in osteogenic medium (OM). (A) First-trimester placenta derived cells aggregate in 7 days and formed nodules at 14-21 days. (B) First-trimester placenta derived cells exhibit alkaline phosphatase activity at 2 weeks. (C) Von Kossa staining-positive aggregates or nodules are found after 3 weeks, which shows significant calcium deposition in osteogenic media.

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  Fig. 5 Adipogenic differentiation from placenta derived cells. Adipogenic differentiation was evaluated by culturing placenta-derived cells in adipogenic induction and maintenance medium for 3 weeks and evidenced by the formation of lipid vacuoles using Oil-Red-O staining and hematoxylin counterstaining. The first-trimester placentaderived cells form lipid vacuoles at 21 day (A-F). The term placenta-derived cells formed lipid vacuoles at 21 day as well (G-L). A, B, G, H: unstained. Original magnification: A, C, G, I, ×40; B, D, H, J ×100; E, K ×200; F, L, ×400.

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  Fig. 6 The expression of Oct4 in first trimester, term placenta and BM derived Mesenchymal stem cells (MSCs). The expression of Oct-4 is strong in first-trimester hPC MSCs and weak in term placenta hPC and adult BM derived MSCs as shown by RT-PCR.

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  Fig. 7 The detection of telomerase activity by the TRAP assay shows the specific signal intensity of telomerase-specific 6-base repeat ladder. First lane shows the expression of telomerase activity in positive control 293T cell; 1st -trimester hPC lane shows the expression of telomerase activity; term hPC and BM MSC lane show the negative telomerase activity.

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  Fig. 8 The detection of pluripotency marker. All of the immunocytochemical stains for SSEA-4, TRA-1-60, and TRA-1-81 on hESCs are strongly positive in group II from first-trimester hPC but very faint in group I (term hPC derived) and group III (BM derived) hMSCs. All magnification: ×40.

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