Figure 3
Figure 3. BM homing of WT hematopoietic progenitor cells (HPCs) is impaired in myeloablated OB/P Cx43-deficient mice. Impaired migration of HSC/Ps through irradiated Cx43-deficient stromal cell lines in a vitro assay. (A) Homing of BM CFU-C in the BM of either irradiated control (empty bar) or OB/P Cx43-deficient (solid bar) recipient mice at 16 hours after transplantation. A dramatic reduction in the ability of HPCs to home into the BM was seen when Cx43 is not expressed in the HM. (B) HSC homing assay in WT or OB/P Cx43-deficient mice. Graph represents homing (%) 3 hours after transplantation of CRUs as analyzed 20 weeks after transplantation into congenic recipients. The secondary recipients were injected with BM cells from primary WT or OB/P Cx43 deficient recipient mice that were previously transplanted with WT 25 × 106 CD45.1+ BM cells. Harvesting of BM cells from primary recipients was performed 3 hours after transplant. (C) Similarly to OB/P Cx43-deficient mice, homing of WT CFU-C to the BM of polyI:C treated Mx1-Cre-Cx43flox/flox mice was drastically reduced compared with homing of either WT or Cx43-deficient HSC/P into Mx1-Cre;WT mice at 16 hours after transplantation. Data represent n = 4 independent experiments with 5 mice per group and experiment. (D) Experimental scheme of the transstromal migration assay. LDBM cells were incubated on a monolayer of BM stromal cells previously layered on a transwell membrane. A Cxcl12 gradient was established from bottom to top of the transwell. (E-F) Transstromal migration of WT (E) or Cx43-deficient (F) HSC/Ps through WT or Cx43-deficient stromal cells. Migration of WT or Cx43-deficient HSC/Ps was similarly impaired when assayed in presence of WT or Cx43-deficient stromal cells. (G) Transstromal migration was restored on reintroduction of rat-Cx43 expression (gray bar) into Cx43-deficient stromal cell lines. Results from WT controls (empty bars) and mock-transduced, Mx1-Cre;Cx43-deficient stromal cells (solid bar) are also depicted. Data for homing experiments are shown as mean ± SEM of 3 independent experiments, with a minimum of 7 mice per group. Data for transstromal migration are shown as mean ± SEM n = 4 independent experiments (*P < .05; **P < .01; ***P < .001; #P = .08).

BM homing of WT hematopoietic progenitor cells (HPCs) is impaired in myeloablated OB/P Cx43-deficient mice. Impaired migration of HSC/Ps through irradiated Cx43-deficient stromal cell lines in a vitro assay. (A) Homing of BM CFU-C in the BM of either irradiated control (empty bar) or OB/P Cx43-deficient (solid bar) recipient mice at 16 hours after transplantation. A dramatic reduction in the ability of HPCs to home into the BM was seen when Cx43 is not expressed in the HM. (B) HSC homing assay in WT or OB/P Cx43-deficient mice. Graph represents homing (%) 3 hours after transplantation of CRUs as analyzed 20 weeks after transplantation into congenic recipients. The secondary recipients were injected with BM cells from primary WT or OB/P Cx43 deficient recipient mice that were previously transplanted with WT 25 × 106 CD45.1+ BM cells. Harvesting of BM cells from primary recipients was performed 3 hours after transplant. (C) Similarly to OB/P Cx43-deficient mice, homing of WT CFU-C to the BM of polyI:C treated Mx1-Cre-Cx43flox/flox mice was drastically reduced compared with homing of either WT or Cx43-deficient HSC/P into Mx1-Cre;WT mice at 16 hours after transplantation. Data represent n = 4 independent experiments with 5 mice per group and experiment. (D) Experimental scheme of the transstromal migration assay. LDBM cells were incubated on a monolayer of BM stromal cells previously layered on a transwell membrane. A Cxcl12 gradient was established from bottom to top of the transwell. (E-F) Transstromal migration of WT (E) or Cx43-deficient (F) HSC/Ps through WT or Cx43-deficient stromal cells. Migration of WT or Cx43-deficient HSC/Ps was similarly impaired when assayed in presence of WT or Cx43-deficient stromal cells. (G) Transstromal migration was restored on reintroduction of rat-Cx43 expression (gray bar) into Cx43-deficient stromal cell lines. Results from WT controls (empty bars) and mock-transduced, Mx1-Cre;Cx43-deficient stromal cells (solid bar) are also depicted. Data for homing experiments are shown as mean ± SEM of 3 independent experiments, with a minimum of 7 mice per group. Data for transstromal migration are shown as mean ± SEM n = 4 independent experiments (*P < .05; **P < .01; ***P < .001; #P = .08).

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