1Institute for Research in Biomedicine, Bellinzona (Switzerland), 2Dipartimento di Biologia e Genetica per le Scienze Mediche, Universitá degli Studi di Milano
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Frascoli, M., Proietti, M., Grassi, F. Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors. J. Vis. Exp. (65), e3736, doi:10.3791/3736 (2012).
The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-α6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.
1. Preparation of Cell Suspension from Bone Marrow
2. Phenotypic Analysis
3. Magnetic Enrichment of c-Kit+ Cells
4. Sorting of Hematopoietic Stem Cells and Lineage committed Progenitors
5. Representative Results
Figure 1 shows and example of the electronic gating procedure to score common lymphoid progenitors (CLPs) as Lin-/c-Kitlo/interleukin (IL)-7R+ cells; Lin-/c-Kit+/Sca-1+ (LKS) and Lin-/c-Kit+/Sca-1lo (c-Kit+) cells; from the c-Kit+ gate, common myeloid progenitors (CMPs) are identified as FcγRloCD34+ cells, granulocyte/monocyte progenitors (GMPs) as FcγRhiCD34+ cells and megakaryocyte/erythrocyte progenitors (MEPs) as FcγRloCD34- cells; from the LKS gate, LT-HSC and ST-HSC are identified as CD34- and CD34+ cells, respectively. This bone marrow cell suspension can be enriched for c-Kit+ cells with magnetic beads (Fig. 2); LT-HSC and ST-HSC can then be sorted according to CD34 expression as well as other progenitors according to the phenotype described above.
HSCs can be analyzed in live imaging confocal microscopy as shown in Figure 4, where CD34- cells were stained with the nucleotide-binding compound quinacrine11 as well as nuclear red (DRAQ5). In HSCs, but not GMPs, quinacrine-positive vesicles are visible in the cytoplasm12. Moreover, sorted HSCs can be used in functional experiments as demonstrated in figure 5, which shows the increase in cytosolic Ca2+ in LT-HSC by activation of purinergic P2 receptors upon exposure to adenosine-triphosphate (ATP) and ionomycin12.
Figure 1. Representative dot plot analysis of Lin- BM cells. Electronic gating procedure at flow cytometer to identify and score hematopoietic progenitors and HSCs from bone marrow cell suspension stained as described in the protocol text.
Figure 2. A scheme for selective enrichment of c-Kit+ cells from bone marrow.
Figure 3. Cell cycle analysis of electronically gated LKS CD34- HSCs in healthy controls and mice with IBD stained with Ki-67 antibody and DAPI. Cells were fixed and permeabilized with Lyse/Fix and Perm buffers followed by staining with FITC conjugated Ki-67 and DAPI at 1 μg/ml. Cycling cells are barely if at all detectable in the LKS CD34- HSCs compartment of healthy controls12.
Figure 4. Live imaging confocal microscopy of FACS sorted CD34- HSCs and GMPs stained with the nucleotide-binding compound quinacrine and nuclear red (DRAQ5). The small quadrants show quinacrine positive vesicles detected in the cytoplasm of HSCs but not GMPs.
Figure 5. Cytosolic Ca2+ elevations in sorted HSCs loaded with Fura-2 and stimulated with ATP and ionomycin. Traces from single cells are shown. All cells were responsive to extracellular ATP showing prominent increase in cytosolic Ca2+ after addition of ATP.
The method described here enables rapid and accurate analysis of hematopoiesis in individual mice (Figure 1). This analysis in various experimental settings, including murine models of inflammation, autoimmunity, immunodeficiency, degenerative diseases, metabolic disorders and cancer, allows addressing the impact of pathological conditions on hematopoiesis. Figure 3 shows the analysis of cell cycling activity on electronically gated LKS CD34- cells from healthy mice and mice with inflammatory bowel disease (IBD)13 by staining with Ki-67 mab and DAPI. The latter displayed a significant increase of cells in S/G2/M phases of the cell cycle. This analysis in flow cytometry demonstrates the impact of T cell mediated inflammation on HSC cycling activity12.
Combination of magnetic labeling and enrichment of bone marrow cells expressing c-Kit with cell sorting in flow cytometry allows isolation of highly purified hematopoietic lineage progenitors and HSCs. The obtained cell populations can be used in a number of assays to study cell biology (Figure 4), signal transduction (Figure 5), developmentally regulated gene expression and in vitro as well as in vivo functional potential of distinct cell subsets. Indeed, magnetically enriched c-Kit+ cells were used to successfully engraft non-irradiated hosts to study dynamic variation of HSCs cycling activity in steady state and inflammation14. In our hands the procedure described here allows more efficient and faster recovery of bone marrow cells than bone flushing with an average yield of 200,000 LKS cells per mouse. The very small number of quiescent HSCs that can be isolated from pooled bone marrows of multiple mice constitutes a limit for experimental approaches requiring more cells. To obviate to this limitation, in many cases preliminary studies can be performed on more abundant LKS cells to envisage more focused and punctual analyses to be performed in CD34- quiescent HSCs.
No conflicts of interest declared.
We thank Nobuyuki Onai, Hitoshi Takizawa and Markus Manz for precious advice. This work was funded by Swiss National Science Foundation, Swiss Cancer League and Fondazione Ticinese per la RIcerca sul Cancro.
|MEM NEAA 100X||Gibco||11140|
|Cell Strainer 40 μm||BD Falcon||352340|
|7-AAD Staining solution||BD Pharmingen||559925|
|Lyse/Fix buffer||BD Pharmingen||558049|
|Perm buffer III||BD Pharmingen||558050|
|Anti-APC MicroBeads||Miltenyi Biotec||130-090-855|
|LS Columns||Miltenyi Biotec||130-042-401|