Mesenchymal Stem Cells (MSCs)
In addition to Hematopoietic Stem Cells, bone marrow contains several progenitor/stem cells that not only play an important role in hematopoiesis, but also have the capacity to differentiate into osteoblasts, adipocytes and chondroblasts. These cells, termed Mesenchymal Stem Cells (MSCs), were first identified as adherent fibroblast-like cells when bone marrow was plated in fetal calf serum containing medium (Fridenshtein, 1982, Arkh Patol.). Originally examined for their critical role in the formation of the hematopoietic microenvironment, MSCs have received additional attention given their ability to form multiple cell types following differentiation. Until recently, it was believed that adult derived stem cells, including MSCs, are restricted in their differentiation potential to lineages of their tissue of origin. However, recent evidence suggests that the bone marrow may contain pluripotent progenitor cells, denoted Multipotent Adult Progenitor Cells (MAPCs). MAPCs, which co-purify with MSCs, have been shown to differentiate into neural cells, skeletal cells, cardiomyocytes, endothelial cells and smooth muscle cells (Jiang, 2002, Nature).
Kits for Mesenchymal Stem Cell Research
Pancreatic Islet Cell Characterization Kit
This Kit (Millipore cat. no. SCR045) provides a convenient set of validated antibodies that allows researchers to reliably identify mature pancreatic islets cells. Along with antibodies generated against discrete hormones secreted by alpha, beta, delta and gamma cells of the pancreatic islets, the kit includes PDX-1 (pancreatic duodenal homeobox gene-1), a master regulator of islet cell development and GLUT-2, a glucose transporter present in beta-islet cells.
Pancreatic Cell Development Pathway Kit
This Kit (Millipore cat. no. SCR046) provides a collection of antibodies that are unique to key transition points along the developmental pathway of pancreatic cells. Included in the kit are antibodies to critical transcription factors expressed during the program of development along with two antibodies to hormones secreted by mature islets cells (FoxA2, Hes-1, Pax 6, IDX-1, Glucagon and Pancreatic Polypeptide).
Pancreatic Cell DTZ Detection Assay
This Kit (Millipore cat. no. SCR047) provides the researcher a simple and quick method to identify insulin-producing beta cells from a mixed cell culture preparation or from pancreatic tissues, by detecting high levels of zinc (typically contained in pancreatic beta cells), with the use of a zinc-chelating agent, DTZ. This kit contains DTZ staining and rinse solutions along with filters and syringes required for live staining reactions.
Mesenchymal Stem Cell Characterization Kit
The Mesenchymal Stem Cell Characterization Kit (Millipore cat. no. SCR018) provides researchers with a convenient means to phenotype mesenchymal stem cells using a panel of antibodies. This kit contains reagents to the MSC markers Integrin ß1, CD54, Collagen type I and Fibronectin, and to the negative markers CD45 and CD14. Also included are mouse and rabbit immunoglobulins for the assessment of background staining.
Rat Mesenchymal Stem Cells express mesenchymal stem cell markers: integrin ß1 (A), CD54 (B), collagen type I (C), and fibronectin (D). Nuclei of the cells were visualized with DAPI (blue). |
Rat Mesenchymal Stem Cell Kit
The Rat Mesenchymal Stem Cell Kit (Millipore cat. no. SCR026) provides ready-to-use primary mesenchymal stem cells isolated from the bone marrow of adult Fisher 344 rats along with a panel of positive and negative selection markers for characterization of the mesenchymal stem cell population. Positive cell markers include antibodies directed against integrin ß1 and CD54, two cell-surface molecules that are present on mesenchymal stem cells (Pittenger, 1999, Science). Negative cell markers include antibodies directed against two specific hematopoietic cell surface markers that are not expressed by mesenchymal stem cells: CD14, present on leukocytes and CD45, present on monocytes and macrophages. Mouse and rabbit immunoglobulins for assessment of background staining are also included.Mesenchymal Stem Cell Adipogenesis Kit
The Mesenchymal Stem Cell Adipogenesis Kit (Millipore cat. no. SCR020) contains reagents that readily differentiate mesenchymal stem cells to an adipogenic lineage as assessed with Oil Red O staining of lipid vacuoles in mature adipocytes. These factors include dexamethasone, IBMX, insulin and indomethacin. Along with Oil Red O staining solution, a hematoxylin solution is provided to counterstain the cell nucleus. Using this kit, we typically obtain > 30% mature adipocytes from the rat bone marrow derived mesenchymal stem cells.
Using the Mesenchymal Stem Cell Adipogenesis Kit, rat mesenchymal stem cells differentiated after 21 days to mature adipocytes as indicated by the accumulation of lipid vacuoles that stain with Oil Red O (A, 10x magnification; B, 40x magnification). Cell nuclei (purple) were stained with Hematoxylin Solution. Control rat skin fibroblast cells did not contain any lipid droplets (data not shown). |
Mesenchymal Stem Cell Osteogenesis Kit
The Mesenchymal Stem Cell Osteogenesis Kit (Millipore cat. no. SCR028) provides a method for differentiating mesenchymal stem cells to an osteoblast phenotype. The kit contains two ECM coating molecules (collagen type I and vitronectin), which have been shown to promote osteogenic differentiation of mesenchymal stem cells (Salasznyk, 2004, J. Biomed. Biotechnol.), and the inducing reagents, dexamethasone, ascorbic acid 2-phosphate and ß-glycerophosphate.Also included is Alizarin Red Solution, a staining solution that is used to detect the presence of calcium in bone. This kit will enable researchers to study the process of osteoblast differentiation in vitro and the effect of growth factors, drugs and toxic agents on bone formation.
Using the Mesenchymal Stem Cell Osteogenesis Kit, rat mesenchymal stem cells readily differentiated to an osteocyte lineage as indicated by Alizarin Red S (ARS) staining (B). ARS staining was not observed in control rat skin fibroblasts that were treated in the same manner (A). Alizarin red S staining demonstrates mineral deposition throughout the culture. |
Mesenchymal Stem Cells (MSCs)
Rat Mesenchymal Stem Cells
Cryopreserved Rat Mesenchymal Stem Cells (Millipore cat. no. SCR027) are ready-to-use primary mesenchymal stem cells isolated from the bone marrow of adult Fisher 344 rats. These cells may be used in conjunction with our characterization and differentiation assays to demonstrate multipotentiality of the starting cell population. Differentiation assays available are the Mesenchymal Stem Cell Adipogenesis Kit (Millipore cat. no.SCR020) and Mesenchymal Stem Cell Osteogenesis Kit (Millipore cat. no.SCR028).Human Pancreatic Mesenchymal Cell Lines
Millipore offers proliferating pancreatic human cell lines that were derived from human fetal pancreatic fibroblasts and provide a convenient and cost-effective alternative to in-house preparation of cell lines. The LT2 Immortalized Pancreatic Mesenchymal Cell line (Millipore cat. no. SCR013) and VIT1 Primary Pancreatic Mesenchymal Cell line (Millipore cat. no. SCR014) are thoroughly characterized by DNA cell cycle analysis, IHC and gene expression profiling.
Also available are specialized media and supplements that have been developed to support optimal growth and cryopreservation of the cells. The LT2 and VIT1 cell lines are expanded using optimized growth media and supplement. The Pancreatic Cell Culture Medium (Millipore cat. no. SCR016) is intended for the preparation of complete cell culture medium in conjunction with Pancreatic Cell Culture Supplement (Millipore cat. no. SCR015). This complete medium is optimized for the culture of LT2 and VIT1 cell lines. Also available is the Pancreatic Cell Cryopreservation Medium (Millipore cat. no. SCR017) for consistent high viability cell freezing.
Advantages
- Proliferating pancreatic human cell lines
- Prolonged and rapid cell growth up to 200 population doublings
- Optimized growth & cryopreservation media
- Validated expansion methods
- Extensively tested and characterized
| Cell Line (Passage number) | Vimentin | CD 90 | Ep-CAM | E-Cadherin | Cytokeratin AE1/AE3 |
| LT2 (passage 7) | + | + | – | – | – |
| VIT1 (passage 5 & 7) | + | + | – | – | – |
| Pancreatic primary | + | + | + | + | + |
Immunohistochemistry results demonstrate that both cell lines express the mesenchymal markers vimentin and CD90 but not common markers of epithelial cells.
Human Neonatal Liver Cell Suspensions
Millipore provides a reliable supply of rare neonate liver cells for research purposes. Neonate donors are an ideal source of human stem/progenitor cells. Tissues procured from adult donors are more plentiful, but the cell populations from adults produce fewer stem cells per gram than younger donors, and these cells are capable of significantly fewer population doublings. Each lot of cells is from a single donor and contains a variety of cell types such as hepatocytes, hepatoblasts, endothelial cells and cholangiocytes.These cells are useful in studies of metabolic diseases such as phenylketonuria in infants, liver transplants, artificial livers and the screening of pharmaceutical drugs.
Primary human liver cells from a 12 day old neonatal donor. Human tissue was obtained in a legal and ethical manner in compliance with federal regulations and guidelines. |
Mesenchymal Stem Cell Growth Factors
To expand MSCs in vitro, it was demonstrated that a combination of mitogenic factors including Platelet Derived Growth Factor [PDGF], Epidermal Growth Factor [EGF], basic Fibroblast Growth Factor [bFGF], Transforming Growth Factor-ß [TGFß] and Insulin Like Growth Factor [IGF] were required (Gronthos, 1995, Blood; Kuznetsov, 1997, J. Bone. Miner. Res.). Alternatively, an MSC culture system that incorporated Platelet Derived Growth Factor-BB [PDGF-BB] in addition to EGF, has been shown to result in a cell doubling time of 48 to 72 hours, and could support purified MSCs for more than 60 population doublings (Reyes, 2001, Blood). An analysis of the culture requirements of MAPCs has shown that a unique combination of growth factors was required for optimal expansion. In addition to EGF and PDGF-BB, murine MAPCs required the addition of Leukemia Inhibitory Factor [LIF] (Jiang, 2002, Nature). Millipore offers many recombinant proteins suitable for the in vitro culture of MSCs.
Mesenchymal Stem Cell Markers
MSCs from various species were originally isolated from bone marrow by their ability to adhere to the surface of the culture vessel (Reyes, 2001, Blood; Pittenger, 1999, Science; Marin, 2002, Exp. Hematol; Kadiyala, 1997, Cell Transplant; Johnstone, 1998, Exp. Cell. Res.; Wakitani, 1995, Muscle Nerve; Berry, 1992, J. Cell. Sci; Mosca, 2000, Clin Orthop). In addition to this methodology, techniques employing panels of monoclonal antibodies have been successfully utilized to define and purify MSCs. However, like many stem cell types, the precise phenotype of MSCs has yet to be determined.
Using monoclonal antibodies to define the expression pattern of cell surface antigens, a number of phenotypes for cultured human MSCs have been reported. These include CD73+, Stro-1+, CD105+, CD34–, CD45– and CD144– (Tuli, 2003, Stem Cells) CD34–, CD44lo, CD45–, CD117–, HLA-I– and HLA-DR– (Reyes, 2001, Blood) and SH2+; SH3+, CD29+, CD44+, CD71+, CD90+, CD106+, CD120a+, CD124+, CD14–, CD34– and CD45-1 (Pittenger, 1999, Science).
While considerable effort has been made to define the pheno type of cultured human MSCs, very little is known about the phenotype of human MSCs in vivo. Using a two-stage process, involving magnetic separation and multiparameter flow cytometry, MSCs were isolated directly from human bone marrow (Jones, 2002, Arthritis and Rhematism) Cells were first purified from bone marrow samples using D7-microbeads, then separated by flow cytometry using a monoclonal antibody to CD45. Sorted cells were uniformly positive for CD105, LNGFR, HLA-DR, CD10, CD13, CD90, Stro-1 and Bone Morphogenic Receptor Type IA [BMPRIA], and negative for CD14, CD34, CD117 and CD133. Furthermore, only cells of this phenotype could proliferate and produce adherent monolayers capable of chondrogenic, osteogenic and adipogenic differentiation.
Similarly, monoclonal antibodies have also been used to characterize murine MSCs of various phenotypes. These include Sca-1+, CD29+, CD44+, c-Kit+, CD105+, CD45–, CD31+, CD34+ (Sun, 2003, Stem Cells) and Sca-1+, CD29+, CD44+, CD81+, CD106+, Nucleostemin+ and CD116–, CD34–, CD45–, CD48–, CD117– and CD135– (Baddoo, 2003, J. Cell. Biochem.) While the phenotype of murine MAPCs within bone marrow remains unknown, cultured MAPCs were phenotypically characterized as being negative for CD34, CD44, CD45, c-Kit, MHC-I and MHC-II. Conversely, these MAPCs express low levels of Flk-1, Sca-1 and Thy-1, and higher levels of CD13 and Stage Specific Embryonic Antigen-1 [SSEA-1]126.
Antibodies for Mesenchymal Stem Cell Differentiation Analysis
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