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Blood, Vol. 112, Issue 12, 4494-4502, December 1, 2008
Heme oxygenase-1 deficiency leads to disrupted response to acute stress in stem cells and progenitors
Blood Cao et al.
112: 4494
Supplemental materials for: Cao et al
Files in this Data Supplement:
- Figure S1. HO activity and HO-1 expression in BM cells under steady-state conditions (JPG, 57.5 KB)
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(A) HO activity in HO-1+/+ and HO-1+/− Lin− BM cells. Lin− BM cells were enriched from whole BM cells using the EasySep™ mouse hematopoietic progenitor enrichment kit (StemCell Technologies). Resulting BM cell population is approximately 1–2% of whole BM cells and highly enriched for HPCs. After cells were sonicated, HO activity was measured according to the protocol described in the Methods and values are displayed as nmoles of CO produced/mg protein (mean ± SD, n=4). (B, C) HO-1 (B) and HO-2 (C) mRNA levels in HPCs as measured by real-time PCR. Lin− BM cells were isolated as described above and total RNA was prepared from these cells. Values are displayed as mean ± SD (n=4). (D, E) Representative FACS plots of the levels of intracellular HO-1 protein. HO-1+/+ and HO-1+/− mice (n=6) were sacrificed and BM cells were harvest and stained with antibodies for phenotypically-defined HSC (CD150+CD48−CD244−), MPP (CD244+CD48−CD150−), and more restricted progenitor (CD48+CD244+CD150−) populations. For analysis of levels of intracellular HO-1 protein, stained cells were fixed, permeabilized, and stained with antibodies against HO-1, and phosphorylated p38MAPK. Levels of HO-1 protein were compared within either HO-1+/+ (D) or HO-1+/− genotype (E).
- Figure S2. The frequency and BrdU incorporation of HSCs and HPCs in HO-1+/− mice under steady-state (JPG, 101 KB)
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(A) Mean frequencies of LT-, ST-HSCs and MPPS are displayed as assessed from c-Kit enriched BM cells (n=3, independent cell isolations of different mice, same below). (B) Frequencies of HPCs and erythroblasts in the BM of HO-1+/− and HO-1+/+ mice. Lin−IL-7R − Sca-1−c-Kit+ cells (LISK) were gated according to their expression of CD34 and Fc R to obtain populations representing CMPs, GMPs and MEPs as described (Akashi et al, 2000). The frequencies of CMPs, GMPs and MEPs in the BM of HO-1+/− and wild-type mice are given as the mean (n=3) percentage of total BM cells or of the LSK subpopulation. Erythroblasts in unfractionated BM cells were measured using CD45 and Ter119. (C) Representative FACS plots of the levels of BrdU incorporation. HO-1+/+ and HO-1+/− mice (n=6) were injected i.p. with 200 µL of BrdU (10 mg/mL) and fed with BrdU (0.8 mg/mL) contained in drinking water for 2 days before sacrificed for BM cell harvest and stained with antibodies for phenotypically-defined HSC (CD150+CD48−CD244−), MPP (CD244+CD48−CD150−), and more restricted progenitor (CD48+CD244+CD150−) populations. Stained cells were fixed and permeabilized and then stained with antibodies against BrdU according to manufacture’s instructions (BD Biosciences).
- Figure S3. Accelerated hematopoietic recovery in HO-1+/− mice treated with 5-FU and in recipients of sufficient number of HO-1+/− BM cells (JPG, 44.7 KB)
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Peripheral blood reticulocyte counts (A) and hemoglobin (Hgb) levels (B) in HO-1+/− or HO-1+/+ mice treated with a single doe of 5-FU (150 mg/kg, i.p.) were performed at different times after treatment. Reticulocyte counts at day 17 were 1,132K ± 477 × 103/µL in HO-1+/− vs. 736K ± 368 × 103/µL in HO-1+/+, respectively, with a p-value of 0.032.
- Figure S4. Accelerated hematopoietic recovery in recipients of sufficient number of HO-1+/− BM cells (JPG, 99.7 KB)
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(A–C) Each lethally irradiated recipients received 5 × 106 whole BM cells and bioluminescence intensities (A), weight (B) and cellularity (C) in the spleens of the recipients of HO-1+/− BM cells were measured at day 30 (n=4, p<0.05, respectively). (D, E) Chimerism analysis of recipients of HO-1+/− GFP+ BM cells demonstrating greater numbers of hematopoietic cells (GFP+) in the peripheral blood, spleen and BM at different time points. GFP expression was acquired by breeding of HO-1+/− mice with a GFP transgenic mouse line. 2 × 106 BM cells from either GFP+HO-1+/− or GFP+HO-1+/+ mice were transferred into lethally-irradiated recipients and donor contribution to hematopoiesis in the peripheral blood, BM, and spleen was analyzed at day 12 (GFP+ cells, n=5, p<0.05, respectively) (D) or in peripheral blood at day 56 (GFP+ CD3+, B220+, Mac-1+, and Gr-1+ cells, n=5, p<0.05, respectively) (E). Mean ± SD values (% of each population in normal GFP+ mice) are displayed for each group.
- Figure S5. HO-1+/− HSCs are capable of homing to and engrafting BM of lethally-irradiated recipients (JPG, 74.7 KB)
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(A) Kaplan-Meier plots displaying compromised ability to provide radioprotection. 3, 6, or 9 × 105 BM cells from HO-1+/− mice were transferred into lethally-irradiated recipients and survivals rates were plotted. (B) Homing ability of HO-1+/− HSCs. 250 luc+ HSCs from either HO-1+/− or HO-1+/+ mice were transferred into lethally-irradiated recipients and images were taken at day 7 after transfer. Images are displayed at the same scale. (C, D) Colony-forming-unit-spleen (CFU-S) derived from 5 × 105 BM cells from HO-1+/− or HO-1+/+ mice were no different either in their size and shape (C) or in their numbers (D). (E) Equivalent competitive capacity of HO-1+/− HSCs to reconstitute irradiated recipients. 750 luc+ (or luc−) HO-1+/+ HSCs were transplanted together with 750 luc− HO-1+/− (or luc+) HSCs into lethally-irradiated recipients and hematopoietic engraftment, as measured by whole body bioluminescent intensity (mean ± SD, photons/sec, n=5, p<0.05), followed over time.
- Figure S6. Decreased numbers of donor-derived erythroid progenitors in recipients of a limited number of HO-1+/− BM cells (JPG, 68 KB)
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(A) The gating strategy for analysis of erythroid progenitor (EP) frequency (Terszowski, G. et al., Blood. 105, 1937–1945). Cells recovered from either BM or spleens of the recipients were first gated for GFP expression (donor marker), and then for the CD71+Ter119−/lo population. EPs were defined as c-Kit+ Sca-1− subset within CD71+Ter119−/lo population. (B) Frequency of the EP population in hematopoietic tissues of recipients. 2 × 106 BM cells from either GFP+HO-1+/− or GFP+HO-1+/+ mice were transplanted into lethally-irradiated recipients (n=3) and donor-derived cells were then recovered for assessment of either donor-derived (GFP+) EP populations (c-Kit+Sca-1− cells in Ter119−CD71+ population) at day 15. Values are mean (± SD, n=3, p<0.05).
- Figure S7. Accelerated exhaustion of HO-1+/− HSCs following repeated hematopoietic insults (JPG, 47.4 KB)
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The fold change of whole body bioluminescence intensities of recipients of serial HO-1+/+ or HO-1+/− HSC transplants.
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