Hematopoietic Stem Cells (HSCs)
Our understanding of bone marrow stem cell biology has been developed over the last 80 years. The development of this field has led to Hematopoietic Stem Cell (HSC) transplants being commonly used for the treatment of hematological malignancies, cancers, as well as in gene therapy studies. Similar to a variety of adult stem cells from other tissues, HSCs exhibit the propensity to differentiate into cells of other lineages. Examples of HSC plasticity include the differentiation of HSCs into muscle cells (Jackson et al., 1999, PNAS), liver cells (Peterson et al., 1999, Science) and multiple cell types (Krause et al., 2001, Cell).
Despite the advances made in our understanding of HSC biology, there are a number of technical barriers associated with the manipulation of HSCs in the laboratory. These include the inability to efficiently expand HSCs ex vivo for therapeutic applications and to accurately identify and sort HSCs from other peripheral blood or bone marrow cells. Millipore provides you with solutions for the purification and expansion of HSCs and for the analysis of their reconstitution activity.
HSC Growth Factors
HSC expansion represents an important challenge for stem cell biology. The successful development of efficient expansion methods would reduce the burden of operative bone marrow transplants for therapeutic applications of this cell type. Additionally, the ability to maintain proliferating HSCs in vitro is critical for gene therapy protocols. Although considerable progress has been made in the improvement of expansion technologies over the last 10 years, the routine application of expanded HSCs in replacement therapy has not occurred. Combinations of growth factors trialed for ex vivo expansion include Flt-3 Ligand, TPO, IL-6 & IL-11 (Lazzari et al., 2001, Br. J. Haematology), IL-3, TPO, SCF and Flt-3 Ligand (Robmanith et al., 2001, Stem Cells), IL-3, GCSF & SCF (Bernstein et al., 1991, Blood), G-CSF and MGDF (Stoney et al., 1996, Exp. Hematology). Recent studies suggest that LIF plays an important role in the development of ex vivo expansion systems for murine and human HSCs. Millipore’s range of cytokines provides researchers with a choice of high quality growth factors for the ex vivo expansion of HSCs.
Hematopoietic Stem Cell Markers
The accurate identification of HSCs using markers expressed on the cell surface has attracted much attention. It is widely accepted that the cell surface glycoprotein CD34 is expressed on cells with hematopoietic potential (Krause et al., 1996, Blood). Consequently, the majority of HSC purification regimes utilize this expression profile. To further fractionate the CD34+ population of cells into lineage committed progenitors and primitive HSCs, additional markers have been used in conjunction with CD34.
A highly purified population of HSCs can be obtained by the depletion of lineage-committed cells from samples using a combination of markers (Lineage negative, or Lin–). More recently, it has been proposed that a distinct set of markers that are closely associated with murine and human HSCs (see Cell Surface Markers table) may improve the accuracy of HSC identification and purification (Weissman, NIH Report, 2001). Lastly, it has been shown that the addition of Flk-2 to the lineage mix allows for the direct isolation of long-term HSCs, with the phenotype c-kit+/lin–/Sca-1+/Flk-2–, from adult murine bone marrow (Christensen and Weissman, 2001, PNAS). Conversely, short-term HSCs were characterized as c-kit+/lin-/Sca-1+/Flk-2+.
The pentaspan glycoprotein AC133 (CD133), first isolated in 1997 (Weigmann et al., 1997, PNAS; Miraglia et al., 1997, Blood), has proven to be an important antigenic marker expressed on human HSCs and progenitor cells (Miraglia et al., 1997, Blood; Yin et al., 1997, Blood). In addition to AC133 antibodies, a monoclonal antibody to the murine orthologue of AC133, Prominin-1 (clone 13A4), has also been shown to be useful for the identification of murine hematopoietic progenitors (Corbeil et al., 2000, J. Biol. Chem). This study showed that mouse Prominin-1 is expressed on a subset of CD34+ progenitor cells isolated from bone marrow, consistent with the expression of the AC133 antigen on human HSCs (Yin et al., 1997, Blood).
Using antibodies to the receptor for the complement molecule C1q (C1qRp/CD93), a novel population of HSCs with hematopoietic and hepatic potential was identified from umbilical cord blood and adult bone marrow (Danet et al., 2002, PNAS). C1qRp was demonstrated to be a positive marker of all bone marrow populating stem cells as it was expressed on both CD34+ and CD34– stem cells. The population of highly purified lineage negative CD45+/CD38–/CD34+or– C1qRp+ cells not only had bone marrow repopulation capacity but could also differentiate into human hepatocytes in vivo.
Cell Surface Markers
| HUMAN HSCs |
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| MOUSE HSCs |
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Hematopoietic Stem Cell Markers
| Marker | Antibodies Available (see product Marker listing for more information) |
HUMAN HEMATOPOIETIC STEM CELL MARKERS HUMAN LINEAGE NEGATIVE MARKERS
It is widely accepted that the cell surface glycoprotein CD34 is expressed on the majority of hematopoietic stem cells. Consequently, the majority of HSC purification regimes utilize this expression profile. To further fractionate the CD34+ population of cells into lineage committed progenitors and primitive HSCs, additional markers have been used. Further improvement in the isolation of HSCs from the mature lineage-committed cells can be achieved by the inclusion of markers expressed by more mature cell lines (lineage negative markers). |
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MURINE HEMATOPOIETIC STEM CELL MARKERS |
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BCRP1 and Mdr1 Markers of Primitive SP Stem Cells
In the absence of a unique cell surface marker, primitive HSCs can be isolated by their ability to efflux fluorescent dyes, such as rhodamine 123 and Hoechst™ 33342 (Spangrude and Johnson, 1990, PNAS; Wolf et al., 1993, Exp. Hematol.) Using flow cytometry, it is possible to isolate a “side population” (SP) of cells that is highly enriched for primitive HSCs (Goodell et al., 1997, Nature Med). This method has been used to successfully isolate murine bone marrow, skeletal and neural derived SP cells (Jackson et al., 1999, PNAS; Hulspas & Quesenberry, 2000, Cytometry). More recently a subpopulation of nestinpositive pancreatic progenitor cells has been found to exhibit a SP phenotype Lechner et al., 2002, Biochem.Biophys. Res Comm.)
It was first demonstrated that the ATP Binding Cassette (ABC) transporter P-glycoprotein (ABCG1), which is encoded by the multidrug resistance (MDR1) gene, is expressed on CD34+ HSCs (Zhou et al., 2000, Nature Medicine; Chandhary & Ronison, 1991, Cell). Overexpression of MDR1 in bone marrow cells leads to an expansion of the SP population, their prolonged survival in culture and an improved ability to repopulate the hematopoietic system of mice following transplantation (Bunting et al., 2000, Blood).
In addition to P-glycoprotein, the membrane transporter Bcrp1 (ABCG2) has also been found to be an important determinant of the SP phenotype.
Differentiation & Reconstitution Analysis
During development and in clinical bone marrow transplantation, HSCs systematically migrate to the bone marrow and other hematopoietic organs by a process referred to as stem cell homing. Stem cells could be detected in functional repopulation assays based on their ability to home to the bone marrow microenvironment and to repopulate transplanted recipients with myeloid and lymphoid cells. A valuable assay for demonstrating the reconstitution activity of human HSCs involves transplanting them into immune deficient NOD/SCID mice (Larochelle et al., 1996, Nat. Med.; Bhatia et al., 1997, PNAS; Gan et al., 1997, Blood). Millipore's panel of antibodies to the human specific pan-leukocyte marker CD45, permits assessment of the multilineage engraftment properties of transplanted human stem cells.
Analysis of HSC Homing & Adhesion
The process of stem cell homing to the microenvironment of the bone marrow is thought to be influenced by a group of signaling molecules known as chemokines.While the physiological role of chemokines as regulators of HSC homing remains unclear, it has been demonstrated that a chemokine, Stromal Cell Derived Factor-1(SDF-1), is important for HSC Marker migration (Whetton & Graham, 1999, Trends Cell Biol.). By binding to CD34+ cells that express the receptor CXCR4, SDF-1 has been shown to be a chemoattractant to the FDCP-mix stem-cell-like cell line (Aiuti et al., 1997, J. Exp. Med.).
Expression of the integrin superfamily of heterodimers plays a significant role in the adhesion of HSCs to the bone marrow micro-environment. Heterodimers expressed by human HSCs include CD49e/CD29 (VLA-5), CD49d/CD29 (VLA-4), CD49f/CD29 (VLA-6) and CD49b/CD29 (VLA-2) (Texido et al., 1992, J. Clin. Invest.). The expression of VLA-4 has been shown to be of functional significance for the homing of HSCs to bone marrow stroma. This was illustrated by the discovery that the homing of murine HSCs to the bone marrow of lethally irradiated recipients was significantly reduced after pretreatment of the donor cells with an antibody to VLA-4 a4 chain (Papayannopoulou et al., 1995, PNAS). In addition to VLA-4, the Leukocyte Function Associated molecule-1 (LFA-1) plays an important role in the interaction of HSCs with bone marrow stroma (Kronenwalt et al., 2000, Stem Cells). Evidence includes the demonstration that circulating CD34+ cells express a lower level of LFA-1 than CD34+ cells residing in the bone marrow (Mohle et al., 1995, Exp. Hematol.).
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