HSC/Ps are retained basally or after G-CSF administration, and located closer to the endosteum in the BM of nonmyeloablated OB/P Cx43-deficient mice. (A) PB CFU-C counts in WT (empty bar) and OB/P Cx43 deficient mice (solid bar). N = 4 independent experiments, n = 5 mice per group. (B) Percentage of chimerism in WT CD45.1⁺ lethally irradiated recipient mice, which were transplanted with a mixture of 5 × 10⁵ WT CD45.1⁺ BM cells (competitor cells) and 400 μL PB from CD45.²⁺ WT or OB/P Cx43-deficient donors. N = 2 independent experiments, n = 5 mice per group. (C) Count of circulating HSC/Ps after G-CSF administration in WT and OB/P Cx43-deficient mice. N = 3 independent experiments, n = 5 mice per group. (D) Hematopoietic progenitor content of BM of G-CSF treated WT and OB/P Cx43-deficient mice. N = 3 independent experiments, n = 5 mice per group. (E) Nonirradiated WT or OB/P Cx43-deficient mice were transplanted with WT BM-EGFP⁺ cells. Percentage of EGFP⁺ LSK and LK⁺S⁻ cell populations, determined by flow cytometry, in the PB 16 hours after transplantation. N = 2 independent experiments, n = 7-8 mice per group. Data are shown as mean ± SEM. (F-G) In vivo imaging using multiphoton microscopy technique of transplanted CFSE-labeled hematopoietic progenitors (Lin⁻c-kit⁺ BM cells average n > 25) clustered closer to the endosteum in OB/P Cx43-deficient mice than in controls (empty circles; n = 2 independent experiments). HSC/Ps were stained with CFSE directly after isolation and transplanted into WT or OB/P Cx43-deficient mice. After 24 hours intravital multiphoton imaging was performed in the tibiae of the recipient animals. (F) HSC/Ps (displayed in green) located distantly from the bone (brown, detected by its SHG signal) in the tibia of a WT recipient. (ii) In addition, the blood vessels could be detected after an intravenous injection of rhodamine-dextran (displayed in red). IVM was performed using a Zeiss LSM-710 microscope with simultaneous detection via external nondescanned detectors and Zeis ZEN software (2009 release). Illumination was performed at 800 or 850 nm using a MaiTai TiSa laser via a 20× water-dipping Ins with 1.0 NA. Images were recorded every 60 seconds. Raw data were reconstructed using Volocity 4.0 software (PerkinElmer/Improvision). The dimensions of the original Z-stack were: X = 85.53 μm; Y = 85.53 μm; and Z = 108 μm. The image resolution (XYZ) was 512 × 512 × 28 μm. The pixel size was 0.167 μm in X and Y and 4 μm in Z. (iii) HSC/Ps (displayed in green) in close contact to the endosteum (brown, detected by its SHG signal) in the tibia of an OB/P Cx43-deficient mouse. (iv) After an injection of rhodamine-dextran also the blood vessels could be detected (displayed in red). The dimensions of the original Z-stack were: X = 227.36 μm and Y = 227.36 μm; Z = 92 μm. Image resolution (XYZ) was 512 × 512 × 28 μm. One pixel in X and Y are equivalent to 0.445 μm and in Z = 4 μm. (G) Distance to endosteum of individual HSC/P analyzed in vivo. Transversal bars denote mean ± SEM (*P < .05; **P < .01).