Mesenchymal stem cells in human placental chorionic villi reside in a vascular Niche
Introduction
The chorionic villi from human term placentae are a rich source of MSCs. The potential utility of PMSCs in therapeutic and regenerative medicine drives current research into their in vitro properties. The (trans)differentiation potential of PMSCs in vitro is now well known but there is scant knowledge of the natural distribution and biology of PMSCs in the chorionic villi of the placenta.
PMSCs are readily isolated from term placentae using a variety of methods. The most popular, routinely used method involves mechanical mincing of the chorionic placental tissue, followed by enzymatic digestion and seeding in stem cell-specific medium [1]. PMSCs selectively attach to the plastic cultureware, proliferate rapidly and are usually prepared without additional enrichment strategies [2]. PMSCs can be differentiated in vitro under specific stimulatory environments into derivatives of the mesenchymal cell lineage such as osteocytes, adipocytes, myocytes and chondrocytes [1], [3], [4], [5], [6], [7]. In addition, there is evidence of differentiation in vitro into cell types characteristic of other lineages such as hepatocyte-like cells and neural-like cells [3], [4], [7], [8], [9], [10], [11], but in vivo evidence for such differentiation is very limited.
Various combinations of cell surface markers are used for the phenotypic characterisation of PMSC in vitro. Common positive markers for PMSCs are also used to identify MSCs from other sources and include CD90 (Thy-1), CD105 (endoglin), CD29 (beta 1 integrin), CD44 (hyaluronic acid), and CD73 (lymphocyte-vascular adhesion protein 2). PMSCs are consistently negative for specific endothelial cell surface markers [e.g. von Willebrand Factor (vWF), CD34 and CD45] and haematopoietic stem cell markers (CD34, CD117).
Despite the extensive phenotypic characterisation of PMSCs in vitro with cell surface markers, there have been no studies to exploit multiple cell surface markers to identify the microenvironment, or niche, of PMSCs in the chorionic villi of placental tissues.
The stem cell niche is a highly balanced microenvironment that allows stem cells to survive and remain quiescent and then respond by replicating, migrating and differentiating to replace or repair tissue when needed [12], [13], [14]. This balance between stem cell proliferation and differentiation is regulated through intracellular integration of a multitude of signals initiated by internal and external stimuli. The identification of the anatomic location of the stem cell niche within the placental chorionic villi not only provides clues as to the nature of these stimuli but can also reveal additional roles for stem cells in placental development and function.
Recent studies have employed a panel of cell surface markers associated with endothelial cells (vWF, CD34), pericytes and endothelial cells (CD146), smooth muscle cells (α-smooth muscle actin), and pericyte-specific antigens (STRO-1 and 3G5) to detect cells with MSC properties in bone marrow, periodontal ligament, dental pulp and human adipose tissue [15], [16], [17], [18]. Importantly, these antibody markers were used to define the MSC niche in these tissues. Furthermore, in these studies the STRO-1, 3G5 and CD146 antibodies were able to enrich for subpopulations of progenitor MSCs [15], [18], [19], [20], [21], [22] that have a high capacity to form fibroblastoid colonies in vitro (called the CFU-F population) [22], which is an important property of stem cells. CFU-F colonies derive from a single stem cell and have the ability to differentiate into many of the cells types that are characteristically derived from the niche [23].
Although STRO-1 and 3G5 antibodies have been used to identify in vitro cultured MSCs derived from the various sources described above, they have not been used for the characterisation of the PMSC niche in the chorionic villi. FACS analysis showed that freshly isolated cells from the placental chorionic villi express STRO-1 and that expression fell to undetectable levels after passage 3 of in vitro culture [3]. Another study however, could not detect STRO-1 expression in PMSCs [2]. 3G5 has not been tested as a cell surface marker for PMSCs and the expression patterns of STRO-1 and 3G5 in the placenta are not known.
Another marker which has been used to enrich the CFU-F population is CD49a [24]. Integrin alpha 1 (CD49a/VLA-1), which is the receptor for laminin and collagen, was also investigated. The CD49a cell surface marker has also been used to identify both human bone marrow mesenchymal stem cells [25] and PMSCs [1].
FACS analysis has been used routinely for the analysis of PMSC populations [1], [3], [4], [5], [6], [7]. In this study, we used cell surface markers, including STRO-1, 3G5, CD146 and CD49a that enrich CFU-F populations, to characterise PMSC preparations by immunohistochemistry. PMSC properties were further demonstrated by CFU-F and differentiation assays. Finally, we used immunohistochemical and immunofluorescence studies to identify the anatomic location of the stem cell niche and to determine differences in the stem cell niche between early and late pregnancy.
Section snippets
Patient details
There were 20 term human placentae collected for this study, with an average patient gestation of 38.5 ± 1.6 weeks (mean ± SD), maternal age of 32.9 ± 6.9 years, baby weight of 3419.0 ± 438.9 g and placental weight of 641.6 ± 90.4 g. Delivery mode was either elective Caesarean section (15/20) or vaginal delivery (5/20) and there were 12/20 males and 8/20 females. All samples were obtained from uncomplicated pregnancies and were therefore considered normal.
First trimester placental tissues were collected
Isolation of stem cells from term placental tissue
Placental stem cells were isolated from term human placentae and grown on untreated plastic cultureware. The mean cell number within a 25 cm2 flask was 9 × 105. These cells were confluent for passaging about every 2 weeks. Stem cell cultures could be passaged up to five times (about 30 population doublings). All experiments in these studies were performed with cells at passage two and each experiment was repeated three or more times.
Western immunoblotting
Western analysis was used to show that STRO-1 and 3G5 were
Discussion
The method of Fukuchi et al. [1], which is used routinely to prepare PMSCs, was employed to prepare cultured cells. Exposing cultured cells to specific media formulations that promote differentiation for 2–3 weeks produced cells that were positive for cell-type specific stains. Differentiation into cells with characteristics of adipocytes, osteocytes and chondrocytes was observed. These PMSCs preparations contained clonogenic cells as revealed by colony forming assays, with a variable frequency
Acknowledgements
We wish to acknowledge the financial support of NHMRC Grant No. 509178, the RWH Foundation, Cecilia Kilkeary Foundation, Eirene Lucas Foundation, Harold & Cora Brennen Benevolent Trust (Equity Trustees), Jack Brockhoff Foundation, J & R McGauran Charitable Trust, Diana Brown Trust (Perpetual Trustees), Helen Macpherson Smith Trust, Thomas R & Rosalinda B Ditchfield Medical Research Endowment Fund and the Wenkart Foundation. Term placental samples were sourced and collected by the research
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