CaMKK2 Knockout Bone Marrow Cells Collected/Processed in Low Oxygen (Physioxia) Suggests CaMKK2 as a Hematopoietic Stem to Progenitor Differentiation Fate Determinant

Abstract

Little is known about a regulatory role of CaMKK2 for hematopoietic stem (HSC) and progenitor (HPC) cell function. To assess this, we used Camkk2-/- and wild type (WT) control mouse bone marrow (BM) cells. BM cells were collected/processed and compared under hypoxia (3% oxygen; physioxia) vs. ambient air (~21% oxygen). Subjecting cells collected to ambient air, even for a few minutes, causes a stress that we termed Extra Physiological Shock/Stress (EPHOSS) that causes differentiation of HSCs and HPCs. We consider physioxia collection/processing a more relevant way to assess HSC/HPC numbers and function, as the cells remain in an oxygen tension closer physiologic conditions. Camkk2-/- cells collected/processed at 3% oxygen had positive and negative effects respectively on HSCs (by engraftment using competitive transplantation with congenic donor and competitor cells and lethally irradiated congenic recipient mice), and HPCs (by colony forming assays of CFU-GM, BFU-E, and CFU-GEMM) compared to WT cells processed in ambient air. Thus, with cells collected/processed under physioxia, and therefore never exposed and naïve to ambient air conditions, CaMKK2 not only appears to act as an HSC to HPC differentiation fate determinant, but as we found for other intracellular mediators, the Camkk-/- mouse BM cells were relatively resistant to effects of EPHOSS. This information is of potential use for modulation of WT BM HSCs and HPCs for future clinical advantage.

Keywords: CaMKK2; CaMKK2 knockout mice; Hematopoietic stem (HSCs) and progenitor (HPCs) cells; Hypoxia cell collection/processing; Phenotypic and functional assessment of HSCs and HPCs.

Fig. 1

Phenotypic analysis of Camkk2−/− bone marrow hematopoietic stem (HSC) and progenitor (HPC) cells isolated under hypoxia then processed under ambient air (21% O2) versus hypoxia (3% O2). In a hypoxic glove box (acclimated to 3% O₂ for 18 hours) BM from femurs of 8 week-old wildtype (WT) or Camkk2−/− mice was flushed in sterile PBS, counted, and split in half so that one half remained under hypoxia and the other half was removed from hypoxia and acclimated to ambient air for 2 h. Nucleated cells were then analyzed by flow cytometry to determine the number of HSCs and HPCs. (A) Long-term (LT)-HSC were defined as Lin⁻ Sca1⁺ cKit⁺ (LSK) Flt3⁻ CD34⁻. (B) Short-term (ST)-HSC were defined as LSK Flt3⁻ CD34⁺. (C) Multipotent progenitors (MPP) were defined as LSK Flt⁺ CD34⁺. (D) Common myeloid progenitors (CMP) were defined as Lin⁻ Sca1⁻ cKit⁺ (LK) FcγII/IIIR^lo CD34⁺. (E) Granulocyte-macrophage progenitors (GMP) were defined as LK FcγII/IIIR^hi CD34⁺. (F) Megakaryocyte-erythrocyte progenitors (MEP) were defined as LK FcγII/IIIR⁻ CD34^−/lo. (A-F) Data are pooled from three independent experiments, 3-4 mice each (n = 9-10 total per group). Stats: two-way ANOVA and post-hoc Tukey’s multiple comparisons. **p < 0.01; ***p < 0.001;****p < 0.0001

Fig. 2

Engraftment advantage for Camkk2−/− HSC/HPC isolated in hypoxia or exposed to ambient air. A-G) WT and/or CaMkk2−/− mice (n = 4 per group; 8 week-old) on a C57BL/6 (CD45.2 + CD45.1−) background were sacrificed and BM was harvested in a 3% O₂, 5% CO₂ chamber (CaMkk2−/− Hypoxia). Half of BM cells from each mouse were split to ambient air 21% O₂ and allowed to equilibrate for 2 h. BM cells from respective donor genotypes were combined to make donor pools. Cells were transplanted by injecting 200,000 whole BM donor cells via tail vein to recipient F1 mice (CD45.2 + CD45.1+). “CaMkk2−/− Hypoxia” cells were injected into recipient mice within the 3% O₂, 5% CO₂ chamber [16], while cells from the “WT Air” and “CaMkk2−/− Air” condition were injected in ambient air. For all mice, a second tail vein injection was then done in ambient air to transplant 200,000 competitor cells, which were BM cells from BoyJ background mice (CD45.2−CD45.1+). A-D) At months 1, 2, 4, and 6 post injection, blood was collected from recipient mice. Red blood cells were lysed, and cells were stained for CD45.2 and CD45.1 cell surface expression and analyzed by flow cytometry. Donor chimerism is shown as a percentage of cells that are CD45.2 + CD45.1− relative to all CD45 expressing cells in the peripheral blood. E-F) At month 6 post-injection, BM cells were harvested from one femur per recipient mouse and cells were stained for donor chimerism (CD45.2 + CD45.1− relative to total BM cells) and myeloid/lymphoid ratio (CD11b+/ sum (B220+ and CD3e+). Stats: 1-way ANOVA and post-hoc Tukey’s multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 3

Nucleated cellularity and in vitro colony formation by wildtype (WT) and CaMkk2−/− bone marrow hematopoietic progenitor cells (HPC) isolated under hypoxia (3% O2) and then plated in ambient air (21% O2) versus hypoxia (3% O2). In a hypoxic glove box bone marrow from the femur of WT or KO mice was harvested in sterile PBS, counted, and split in half so that one half remained under hypoxia and the other half was acclimated to ambient air for 2 hours. Nucleated cells were counted and methylcellulose cultures established to enumerate CFU-GM, BFU-E, and CFU-GEMM. Cells were plated at 5 × 10⁴ nucleated cells per ml containing fetal bovine serum (30% v/v), PWMSCM (5% vol/vol), mSCF (50 ng/ml), erythropoietin (1 U/ml), and Hemin (0.1 mM). Cultures were incubated for 6 days at 5%CO₂/5% oxygen in a humidified environment. All culture reagents used to plate cells remaining in hypoxia were acclimated in the hypoxia chamber for 16-18 h. Results are shown for 2 separate experiments with a total n = 7. (A) Nucleated cellularity, (B) CFU-GM, (C), BFU-E, and (D) CFU-GEMM are shown. Statistical analysis was performed using t-tests (A) or two-way ANOVA with Tukey post hoc tests (B-D). *p < 0.05; **p < 0.01; ***p < 0.001