Selection using the α1-integrin (CD49a) enhances the multipotentiality of the mesenchymal stem cell population from heterogeneous bone marrow stromal cells
ABSTRACT Bone marrow-derived mesenchymal stem cells consist of a developmentally heterogeneous population of cells obtained from colony forming progenitors. As these colonies express the alpha-1 integrin (CD49a), here we single-cell FACS sorted CD49a+ cells from bone marrow in order to create clones and then compared their colony forming efficiency and multilineage differentiation capacity to the unsorted cells. Following selection, 40% of the sorted CD49a+ cells formed colonies, whereas parental cells failed to form colonies following limited dilution plating at 1 cell/well. Following ex vivo expansion, clones shared a similar morphology to the parental cell line, and also demonstrated enhanced proliferation. Further analysis by flow cytometry using a panel of multilineage markers demonstrated that the CD49a+ clones had enhanced expression of CD90 and CD105 compared to unsorted cells. Culturing cells in adipogenic, osteogenic or chondrogenic medium for 7, 10 and 15 days respectively and then analysing them by quantitative PCR demonstrated that CD49a+ clones readily underwent multlineage differentiation into fat, bone and cartilage compared to unsorted cells. These results thus support the use of CD49a selection for the enrichment of mesenchymal stem cells, and describes a strategy for selecting the most multipotential cells from a heterogeneous pool of bone marrow mononuclear stem cells.
- SourceAvailable from: Shaun P Brennecke
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- "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 . Integrin alpha 1 (CD49a/VLA-1), which is the receptor for laminin and collagen, was also investigated. "
ABSTRACT: The chorionic villi of human term placentae are a rich source of mesenchymal stem cells (PMSCs). The stem cell "niche" within the chorionic villi regulates how PMSCs participate in placental tissue generation, maintenance and repair, but the anatomic location of the niche has not been defined. A number of cell surface markers for phenotypic characterisation of mesenchymal stem cells (MSCs) were employed to identify the stem cell niche within the chorionic villi of first trimester and term human placenta. This included antibodies to pericyte cell surface markers STRO-1 and 3G5, which have been used to identify mesenchymal stem cells in other tissues, but have not been studied in placental tissues. PMSCs were isolated from term human placentae and shown to have stem cell properties by their ability to grow on untreated plastic culture ware, capacity for forming clones (i.e. clonogenicity) and their capability to differentiate into adipocytes, chondrocytes and osteocytes. Western analysis confirmed that STRO-1 and 3G5 are present in placental protein extracts and in PMSCs. Immunocytochemistry revealed PMSCs were positive for MSC cell surface markers (STRO-1, 3G5, CD105, CD106, CD146, CD49a, alpha-SMA) and negative for haematopoietic stem cell markers (CD117, CD34) and endothelial markers (CD34, vWF). Immunohistochemistry with antibodies to MSC cell surface markers on first trimester and term tissues revealed a vascular niche for PMSCs. Dual-label immunofluorescence analysis was used to compare STRO-1 antibody staining with that of endothelial cell marker vWF and found no significant overlap in staining. This indicated that some PMSCs have a pericyte-like phenotype. We propose that the vascular niche harbours a pool of PMSCs that can give rise to committed progenitors for tissue maintenance and repair, and that PMSCs contribute to vessel maturation and stabilization.Placenta 03/2010; 31(3):203-12. DOI:10.1016/j.placenta.2009.12.006 · 3.29 Impact Factor
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- "Several attempts for purification of bone marrow-derived MSCs have been made using magnetic beads to select CD49a + [Deschaseaux et al., 2003; Rider et al., 2007], CD105 + [Aslan et al., 2006; Jarocha et al., 2008; Wong et al., 2008] and CD271 + cells [Battula et al., 2008; Jarocha et al., 2008]. To our knowledge, CD90 has not yet been used for the immunomagnetic isolation of ASCs, but is highly expressed. "
ABSTRACT: High hopes are put into the use of mesenchymal stem cells (MSCs) in various approaches for tissue engineering and regenerative medicine. MSCs are derived from different tissues with only small differences in their phenotype or their differentiation potential, but higher differences in the cell yield. Since fat is easily accessible and contains a high amount of MSCs to be isolated, adipose-derived stem cells (ASCs) are very promising for clinical approaches. ASCs are not a completely homogeneous cell population. Our study was initiated to explore an easy and convenient method to reduce heterogeneity. We tested different isolation methods: (1) the standard isolation method for ASCs based on plastic attachment, (2) the standard method with an initial washing step after 60 min of adherence and (3) immunomagnetic isolation by 4 typical markers (CD49a, CD90, CD105 and CD271). Cells isolated by these methods were evaluated using quantitative PCR and flow cytometry as well as by their differentiation potential. Washing led to a significantly lower expression of desmin, smA and six2, and a higher expression of the stem cell markers nestin, oct-4 and sall-1, compared to standard isolated cells, while the immunomagnetically isolated cells showed no significant changes. All cells independent of the isolation method could be induced to differentiate into adipocytes and osteoblasts. Our study demonstrates that a simple washing step reduces heterogeneity of cultured ASCs according to PCR analysis, whereas the immunomagnetic isolation only showed minor advantages compared to the standard method, but the disadvantage of significantly lower cell yields in the primary isolates.Cells Tissues Organs 02/2010; 192(2):106-15. DOI:10.1159/000289586 · 2.14 Impact Factor
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ABSTRACT: Mesenchymal stem cells (MSCs) hold great promise for the application of tissue engineering to orthopedic problems such as critical-size defects. Cell sorting has indicated a benefit to enriching the MSC population, but is undesired for clinical applications. Pharmacological enrichment utilizing adipogenic inhibitors such as resveratrol represent a clinically viable alternative. Cells were isolated from rats, and treated with differentiation media to verify multipotency, and were then treated with 0, 12.5, and 25 µM resveratrol in growth media and osteogenic media. MSC and osteoprogenitor (OPC) populations were measured using flow cytometry and OPC quality was assessed with osteocalcin production, osteoprotegerin production, and alkaline phosphatase activity. Resveratrol increased the population percent and cell number of MSCs in both growth and osteogenic media, but only increased the number of OPCs in growth media. In both media types resveratrol increased alkaline phosphatase activity and osteocalcin levels. Resveratrol enriches ADMSCs for mesenchymal stem cells and osteoprogenitors. Boyan, Barbara - Faculty Mentor ; Guldberg, Robert - Committee Member/Second Reader