Immunohistochemical Localization and Characterization of Putative Mesenchymal Stem Cell Markers in the Retinal Capillary Network of Rodents

The tissue-engineered heart valve may be the ideal valve replacement option but still must overcome challenges in leaflet recellularization. This study sought to investigate the potential for leaflet matrix restoration and repopulation following mononuclear cell seeding and extended periods of bioreactor conditioning. Human aortic heart valves were seeded with mononuclear cells and conditioned in a pulsatile bioreactor for 3 days, 3 weeks, or 6 weeks. The results of this study determined that a mononuclear cell population can be readily localized within the leaflet tissue in as little as 3 days. Furthermore, as extended bioreactor condition continued to the 3- and 6-week time points, the mesenchymal stem cell subfraction proliferated and appeared to become the predominant cell phenotype. This was evident through positive expression of mesenchymal stem cell markers and no expression of mononuclear cell markers observed by immunohistochemistry in the 3- and 6-week groups. In addition, cells in the 3- and 6-week groups exhibited an up-regulation of mesenchymal stem cell–associated genes ( THY1, NT5E, and ITGB1) and a down-regulation of mononuclear cell–associated genes ( CD14, ICAM1, and PECAM1) compared to the initial seeded cell population. However, repopulation of the leaflet interstitium was less extensive than anticipated. Valves in the 6-week time point also exhibited retracted leaflets. Thus, while the 3-week bioreactor-conditioning period used in this study may hold some promise, a bioreactor-conditioning period of 6 weeks is not a viable option for clinical translation due to the negative impact on valve performance.

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Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells

PeerJ ◽

10.7717/peerj.3180 ◽

2017 ◽

Vol 5 ◽

pp. e3180 ◽

Cited By ~ 2

Author(s):

Rohaya Megat Abdul Wahab ◽

Nur Akmal Mohamed Rozali ◽

Sahidan Senafi ◽

Intan Zarina Zainol Abidin ◽

Zaidah Zainal Ariffin ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Dental Pulp ◽

Doubling Time ◽

Enzymatic Digestion ◽

Stem Cell Markers ◽

Isolation Method ◽

Cell Markers

Background Stem cells are normally isolated from dental pulps using the enzymatic digestion or the outgrowth method. However, the effects of the isolation method on the quality of the isolated stem cells are not studied in detail in murine models. The aim of this study was to compare the matrices secreted by osteoblast and chondrocytes differentiated from dental pulp stem cells isolated through different means. Method DPSC from murine incisors were isolated through either the outgrowth (DPSC-OG) or the enzymatic digestion (DPSC-ED) method. Cells at passage 4 were used in this study. The cells were characterized through morphology and expression of cell surface markers. The cells’ doubling time when cultured using different seeding densities was calculated and analyzed using one-way ANOVA and Tukey’s multiple comparison post-test. The ability of cells to differentiate to chondrocyte and osteoblast was evaluated through staining and analysis on the matrices secreted. Results Gene expression analysis showed that DPSC-OG and DPSC-ED expressed dental pulp mesenchymal stem cell markers, but not hematopoietic stem cell markers. The least number of cells that could have been used to culture DPSC-OG and DPSC-ED with the shortest doubling time was 5 × 102 cells/cm2 (11.49 ± 2.16 h) and 1 × 102 cells/cm2 (10.55 h ± 0.50), respectively. Chondrocytes differentiated from DPSC-ED produced 2 times more proteoglycan and at a faster rate than DPSC-OG. FTIR revealed that DPSC-ED differentiated into osteoblast also secreted matrix, which more resembled a calvaria. Discussion Isolation approaches might have influenced the cell populations obtained. This, in turn, resulted in cells with different proliferation and differentiation capability. While both DPSC-OG and DPSC-ED expressed mesenchymal stem cell markers, the percentage of cells carrying each marker might have differed between the two methods. Regardless, enzymatic digestion clearly yielded cells with better characteristics than outgrowth.

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