Hematopoietic stem cells are uniquely selective in their migratory response to chemokines

Abstract

Although hematopoietic stem cell (HSC) migration into and out of sites of active hematopoiesis is poorly understood, it is a critical process that underlies modern clinical stem cell transplantation and may be important for normal hematopoietic homeostasis. Given the established roles of chemotactic cytokine (chemokine)-directed migration of other leukocyte subsets, the migration of murine HSC to a large panel of CC and CXC chemokines was investigated. HSC migrated only in response to stromal derived factor-1alpha, the ligand for the CXC chemokine receptor 4 (CXCR4). CXCR4 expression by HSC was confirmed by reverse transcription polymerase chain reaction analysis. Surprisingly, HSC also expressed mRNA for CCR3 and CCR9, although they failed to migrate to the ligands for these receptors. The sharply restricted chemotactic responsiveness of HSC is unique among leukocytes and may be necessary for the specific homing of circulating HSC to bone marrow, as well as for the maintenance of HSC in hematopoietic microenvironments.

Figure 1.

LT-HSC and ST-HSC migrate in response to SDF-1α, but are refractory to other chemokines and to G-CSF. (A) Flow cytometry contour plots showing partial enrichment of BM cells for HSC by lineage depletion (upper two panels), and gating of lineage⁻ LT-HSC (boxed cells, lower left) and lineage^lo ST-HSC (boxed cells, lower right). See the Materials and Methods section for details. (B and C) Lineage-depleted BM cells were prepared and added to inserts placed in wells containing medium alone, the listed chemokines, or G-CSF (see the Materials and Methods section for concentrations). Responding cells were harvested, stained for HSC markers, and analyzed for the presence and number of (B) LT-HSC and (C) ST-HSC by flow cytometry. The data presented are the means ±SD of two to nine independent experiments and represent the percentage of input HSC that migrated to each chemokine or to G-CSF. Numbers in parentheses indicate the number of experiments performed for each agent. CC and CXC refer to the receptor families to which the listed chemokines belong. Compared with basal migration, only migration to SDF-1α was statistically significant (P < 0.05).

Figure 1.

LT-HSC and ST-HSC migrate in response to SDF-1α, but are refractory to other chemokines and to G-CSF. (A) Flow cytometry contour plots showing partial enrichment of BM cells for HSC by lineage depletion (upper two panels), and gating of lineage⁻ LT-HSC (boxed cells, lower left) and lineage^lo ST-HSC (boxed cells, lower right). See the Materials and Methods section for details. (B and C) Lineage-depleted BM cells were prepared and added to inserts placed in wells containing medium alone, the listed chemokines, or G-CSF (see the Materials and Methods section for concentrations). Responding cells were harvested, stained for HSC markers, and analyzed for the presence and number of (B) LT-HSC and (C) ST-HSC by flow cytometry. The data presented are the means ±SD of two to nine independent experiments and represent the percentage of input HSC that migrated to each chemokine or to G-CSF. Numbers in parentheses indicate the number of experiments performed for each agent. CC and CXC refer to the receptor families to which the listed chemokines belong. Compared with basal migration, only migration to SDF-1α was statistically significant (P < 0.05).

Figure 1.

LT-HSC and ST-HSC migrate in response to SDF-1α, but are refractory to other chemokines and to G-CSF. (A) Flow cytometry contour plots showing partial enrichment of BM cells for HSC by lineage depletion (upper two panels), and gating of lineage⁻ LT-HSC (boxed cells, lower left) and lineage^lo ST-HSC (boxed cells, lower right). See the Materials and Methods section for details. (B and C) Lineage-depleted BM cells were prepared and added to inserts placed in wells containing medium alone, the listed chemokines, or G-CSF (see the Materials and Methods section for concentrations). Responding cells were harvested, stained for HSC markers, and analyzed for the presence and number of (B) LT-HSC and (C) ST-HSC by flow cytometry. The data presented are the means ±SD of two to nine independent experiments and represent the percentage of input HSC that migrated to each chemokine or to G-CSF. Numbers in parentheses indicate the number of experiments performed for each agent. CC and CXC refer to the receptor families to which the listed chemokines belong. Compared with basal migration, only migration to SDF-1α was statistically significant (P < 0.05).

Figure 4.

The magnitude of the chemotactic response to SDF-1α by HSC from BM of untreated mice is indistinguishable from the magnitude of the chemotactic responses of HSC derived from BM, blood, or spleens of Cy/G-CSF–treated mice. Lineage-depleted BM cells from untreated animals, or BM, blood, or spleen cells from day 4 Cy/G-CSF–treated animals were prepared and added to inserts placed in wells containing medium alone or SDF-1α. Responding cells were harvested, stained for HSC markers, and analyzed for the presence and number of LT-HSC and ST-HSC by flow cytometry. The data presented are the means ±SD of four to nine independent experiments and represent the percentage of input HSC that migrated. Numbers in parentheses indicate the number of experiments performed. Shaded bar, LT-HSC; filled bar, ST-HSC.

Figure 2.

(A) HSC migration to SDF-1α is chemotactic, not chemo-kinetic. Lineage-depleted BM cells were prepared and added to inserts placed in wells. SDF-1α was present in either the bottom well, the top well (the insert), or in both the top and bottom wells. Responding cells were harvested, stained for HSC markers, and analyzed for LT-HSC and ST-HSC by flow cytometry. The data represent the percentage of input HSC that migrated to the bottom well. HSC migrated only in the presence of a gradient of increasing SDF-1α concentration. (B) HSC chemotaxis to SDF-1α does not require cells other than HSC. 15,000 Thy-1.1^loSca-1⁺Lin^−/loc-Kit⁺ cells (this population contains both LT-HSC and ST-HSC) from BM of untreated mice were sorted directly into inserts placed in wells containing SDF-1α. 30,000 of the same cells were sorted into inserts placed in wells only containing medium to establish basal migration. Responding cells were collected and analyzed by flow cytometry. The data represent the percentage of input HSC that migrated to the bottom well.

Figure 2.

(A) HSC migration to SDF-1α is chemotactic, not chemo-kinetic. Lineage-depleted BM cells were prepared and added to inserts placed in wells. SDF-1α was present in either the bottom well, the top well (the insert), or in both the top and bottom wells. Responding cells were harvested, stained for HSC markers, and analyzed for LT-HSC and ST-HSC by flow cytometry. The data represent the percentage of input HSC that migrated to the bottom well. HSC migrated only in the presence of a gradient of increasing SDF-1α concentration. (B) HSC chemotaxis to SDF-1α does not require cells other than HSC. 15,000 Thy-1.1^loSca-1⁺Lin^−/loc-Kit⁺ cells (this population contains both LT-HSC and ST-HSC) from BM of untreated mice were sorted directly into inserts placed in wells containing SDF-1α. 30,000 of the same cells were sorted into inserts placed in wells only containing medium to establish basal migration. Responding cells were collected and analyzed by flow cytometry. The data represent the percentage of input HSC that migrated to the bottom well.

Figure 3.

LT-HSC and ST-HSC contain mRNA for CCR3, CCR9, and CXCR4. (A) RT-PCR analysis of mRNA for chemokine receptors of combined LT-HSC and ST-HSC (Thy-1.1^loSca-1⁺Lin^−/loc-Kit⁺ cells). RT-PCR was performed on RNA isolated from the equivalent of 1,000 double-sorted HSC, or from 1,000 unfractionated WBM cells. Representative data are shown (see Table IV for data summary). (B) Additional RT-PCR was performed on mRNA isolated from the equivalent of 1,000 LT-HSC or 1,000 ST-HSC for the receptors found to be positive in the first screen. Both LT-HSC and ST-HSC contained mRNA for CXCR4, CCR3, and CCR9. See the Materials and Methods section for RT-PCR protocol. DHFR, dihydrofolate reductase; WBM, whole bone marrow.

Figure 3.

LT-HSC and ST-HSC contain mRNA for CCR3, CCR9, and CXCR4. (A) RT-PCR analysis of mRNA for chemokine receptors of combined LT-HSC and ST-HSC (Thy-1.1^loSca-1⁺Lin^−/loc-Kit⁺ cells). RT-PCR was performed on RNA isolated from the equivalent of 1,000 double-sorted HSC, or from 1,000 unfractionated WBM cells. Representative data are shown (see Table IV for data summary). (B) Additional RT-PCR was performed on mRNA isolated from the equivalent of 1,000 LT-HSC or 1,000 ST-HSC for the receptors found to be positive in the first screen. Both LT-HSC and ST-HSC contained mRNA for CXCR4, CCR3, and CCR9. See the Materials and Methods section for RT-PCR protocol. DHFR, dihydrofolate reductase; WBM, whole bone marrow.