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    • The presence of multi lineage

    2018-10-29

    The presence of multi-lineage mesenchymal stem SB431542 in the placenta has tremendous implications. Along with the multi-differentiation capability, the easy accessibility, the abundant source and no ethical concerns, placenta-derived MSCs (PMSCs) may be an attractive, alternative progenitor or stem cells for basic research and clinical applications (Winkler et al., 2005). However, the question arises as how to isolate and purify efficiently PMSCs from placenta. In the previous research, multi-lineage mesenchymal stem cells from placenta were isolated generally using the direct attachment method (Miao et al., 2006; Soncini et al., 2007; Huang et al., 2009; Dimitrov et al., 2010; Fukuchi et al., 2004). The direct attachment method used in various reports was different. Dimitrov et al. (2010) reported that cells adhered in 24h could be isolated into PMSCs. However, Miao et al. (2006) and Fukuchi et al. (2004) found that cells needed 3–5d of initiating incubation before the adherent cells could be cultured into cell colonies. It may imply that the isolation of multi-lineage mesenchymal stem cells from placenta tissue should not be simple as the isolation of those cells from bone marrow. Our results showed that MSC-like cells in the primary passage from placental tissue should need a longer time to adhere, and cells isolated using the time-gradient attachment for 72h were easier to expand. Although the direct attachment method is an easier technique to isolate MSCs from various tissues, the cell population isolated from placenta using this method in our lab is always an impure population incorporated with other fibroblast-like cells. Therefore, it is necessary to use a technique effective enough to purify MSC-like cells and get rid of other fibroblast-like cells. In order to solve this key problem, the step-by-step isolated method was developed (Miki et al., 2007; Takashima et al., 2004; Wei et al., 2003; Portmann-Lanz et al., 2006; Wolbank et al., 2007; Bailo et al., 2004; Ilancheran et al., 2007). Mechanical peeling of amnion membrane from the underlying chorion was followed by digestion with dispase II and then trypsin–EDTA. In this isolation system, cells in tissue undergo a long-term of digestion, which may damage cells. As a source of MSC-like cells, the placenta also contains some cells with the adherent characteristics similar to MSCs, such as vascular endothelial cells or fibroblast cells (Miki et al., 2005; Richards et al., 1995; Markoff et al., 1983; Terada et al., 2000; Bilic et al., 2004). However, the ability of adhesion between different cells from placenta was various. A preplate culture method has been developed to isolate MSC-like cells form muscle (Gharaibeh et al., 2008; Clause et al., 2010; Ji et al., 2010; Sharifiaghdas et al., 2011). This technique is used to select cells after serial adherences, based on their special adherence ability to plastic. We modified this method as the time-gradient attachment method (see Materials and methods) to isolate and expand PMSCs from human term placentas. We digested cells from the decidua basalis that contained abundant MSC-like cells (In\'t Anker et al., 2004) and isolated PMSCs using the direct attachment and the time-gradient attachment. The results showed that cells isolated using both attachment methods could be expanded in vitro. These cells were fibrous morphology and the proliferation potential remained after enzymatic digestion and passaging. However, cells isolated using the time-gradient attachment for 72h had the higher proliferation potential in comparison with cells isolated using the direct attachment, especially in the later passages. Meanwhile, the phenotype profile of cells isolated using the direct attachment was positive for CD29, CD73, CD90, CD105 and CD117, and negative for CD34 and CD45. The phenotype profile of cells isolated using the time-gradient attachment for 72h was positive for CD29, CD73, CD90 and CD105, and negative for CD 34, CD45 and CD117. It is a generally consensus that MSC-like cells are negative for CD34, CD45 and CD117 (Baksh et al., 2003). CD117, a c-kit proto-oncogene product was observed both in the spindle cell and epithelioid subtypes (Sarlomo-Rikala et al., 1998). Therefore, it could be concluded that cells isolated from placenta were a complex population that consisted of various cells with different adhesion abilities and that cells isolated using the direct attachment might contain other cells, such as epithelioid cells. In addition, the analysis of multi-differentiation potentials confirmed that cells isolated using the time-gradient attachment for 72h should have the higher multi-differentiation potentials. Therefore, our results proved that the cells isolated using the time-gradient attachment method should be MSC-like cells. It suggested that the time-gradient attachment method could be a more practicable and reproducible method for isolation of PMSCs from placenta. The most significant concern is that although cells derived using the direct attachment method here had also characteristics as bona fide MSC cells that had previously derived using a similar method (Huang et al., 2009; Dimitrov et al., 2010; Fukuchi et al., 2004), cells isolated using the time-gradient attachment for 72h should contain more PMSCs than cells isolated using the direct attachment in this study.