J Adv Prosthodont.  2014 Oct;6(5):351-360. 10.4047/jap.2014.6.5.351.

Characteristics and response of mouse bone marrow derived novel low adherent mesenchymal stem cells acquired by quantification of extracellular matrix

• Zheng RC
• Kim SK
• Heo SJ
• Koak JY
• Lee JH
• Park JM

• KMID: 2284735
• DOI: http://doi.org/10.4047/jap.2014.6.5.351

Abstract

PURPOSE
The aim of present study was to identify characteristic and response of mouse bone marrow (BM) derived low-adherent bone marrow mesenchymal stem cells (BMMSCs) obtained by quantification of extracellular matrix (ECM).
MATERIALS AND METHODS
Non-adherent cells acquired by ECM coated dishes were termed low-adherent BMMSCs and these cells were analyzed by in vitro and in vivo methods, including colony forming unit fibroblast (CFU-f), bromodeoxyuridine (BrdU), multi-potential differentiation, flow cytometry and transplantation into nude mouse to measure the bone formation ability of these low-adherent BMMSCs. Titanium (Ti) discs with machined and anodized surfaces were prepared. Adherent and low-adherent BMMSCs were cultured on the Ti discs for testing their proliferation.
RESULTS
The amount of CFU-f cells was significantly higher when non-adherent cells were cultured on ECM coated dishes, which was made by 7 days culturing of adherent BMMSCs. Low-adherent BMMSCs had proliferation and differentiation potential as adherent BMMSCs in vitro. The mean amount bone formation of adherent and low-adherent BMMSCs was also investigated in vivo. There was higher cell proliferation appearance in adherent and low-adherent BMMSCs seeded on anodized Ti discs than machined Ti discs by time.
CONCLUSION
Low-adherent BMMSCs acquired by ECM from non-adherent cell populations maintained potential characteristic similar to those of the adherent BMMSCs and therefore could be used effectively as adherent BMMSCs in clinic.

Keyword

Bone marrow mesenchymal stem cells; Quantification; Low-adherent; Non-adherent cells; Extracellular matrix; Bone formation

MeSH Terms

Animals
Bone Marrow*
Bromodeoxyuridine
Cell Proliferation
Extracellular Matrix*
Fibroblasts
Flow Cytometry
Mesenchymal Stromal Cells*
Mice*
Mice, Nude
Osteogenesis
Stem Cells
Titanium
Bromodeoxyuridine
Titanium

Figure

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  Fig. 1 The picture shows the scheme for obtaining low-adherent BMMSCs on an ECM coated dishes.

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  Fig. 2 Ability of the ECM coated dishes to obtain low-adherent MSCs in non-adherent cell populations after primary culture of mouse bone marrow mononuclear cells. Primary all mononuclear cells were seeded and incubated for 48 hours under 37℃ at 5% CO2, and then the non-adherent cells were gathered. Non-adherent cell suspensions were replated on the ECM coated dishes made by 1, 4, 7, 10 days incubation of MSCs in standard medium under 37℃ at 5% CO2 condition. The cultures were maintained as described for the determination of CFU-f number. CFU-f on the extracellular matrix coated dishes made by 1 day (A), 4 days (B), 7 days (C), 10 days (D) incubation. Original magnification ×100. The number of CFU-f (E).

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  Fig. 3 SEM images of an ECM coated dishes by day; 1 day (A, E), 4 days (B, F), 7 days (C, G), 10 days (D, H). Upper panels showed the ECM coated dishes made by cultured adherent bone marrow mesenchymal stem cells after cell decellularized at low magnification (×100). Low panels showed at high magnification (×500).

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  Fig. 4 CFU-f assays. Primary all nuclear cells (ANCs) were seeded on plastic culture dishes and incubated for 48 hours under 37℃ at 5% CO2 condition, and then the non-adherent cell suspensions were collected from plastic cultured dishes. Non-adherent cells were reseeded on ECM coated culture dishes and incubated for 14 days in standard medium under 37℃ at 5% CO2 condition. After 14 days, CFU-f staining was performed. The colony numbers were significantly increased on the extracellular matrix coated dishes. Colony image of adherent BMMSCs on plastic culture dish (A, B). Colony image of non-adherent cells on ECM coated dish (C, D). Original magnification: ×200 (B, D). The number of CFU-f (F).

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  Fig. 5 The adherent and low-adherent BMMSCs proliferation rate was measured by BrdU assay. (A) BrdU images of adherent BMMSCs, Original magnification: ×200. (B) BrdU images of low-adherent MSCs, ×200. (C) The BrdU uptake percentage of positive cells.

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  Fig. 6 Bone nodule formation ability assessed by Alizarin Red S staining were not significantly different between adherent BMMSCs (A, B) and low-adherent BMMSCs (C, D). The percentage of bone nodule formation (E). Runx2 (293) was expressed in osteogenic cultures of both adherent BMMSCs and low-adherent BMMSCs (F).

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  Fig. 7 Oil cluster formation ability assessed by Oil red O staining was not significantly different between adherent BMMSCs (A, B, C) and low-adherent BMMSCs (D, E, F). The percentage of the Oil red O positive cells (G). LPL (365 bp) and PPAR-2 (352 bp) were expressed in adipogenic cultures of both adherent BMMSCs and low-adherent BMMSCs (H).

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  Fig. 8 Flow cytometric analysis of adherent BMMSCs and low-adherent BMMSCs were analyzed by CD29, CD44, Oct-4, CD14, CD34, and CD117. The CD14, CD34, and CD117 are positive stem cell markers (A) and CD29, CD44, and Oct-4 are negative stem cell markers (B). The percentage of positive cells (C).

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  Fig. 9 Sections were harvested at 8 week and stained with H&E, (A) Adherent BMMSCs, (B) Low-adherent BMMSCs. The percentage of the bone formation (C).

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  Fig. 10 SEM images of adherent BMMSCs and Low-adherent BMMSCs seeded on Ti discs. Upper panels show the adherent BMMSCs seeded on machined discs (A, B, C). Low panels show the low-adherent BMMSCs on machined discs (D, E, F) SEM images of adherent BMMSCs and low-adherent BMMSCs on anodized discs. Upper panels showed the adherent BMMSCs (G, H, J) seeded on anodized discs. Low panels showed the low-adherent BMMSCs (J, K, L) seeded on anodized discs. The proliferation rates of adherent BMMSCs and low-adherent BMMSCs seeded on the machined and anodized discs for 1, 4, 7 days (M).

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