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Translational and Clinical Research
Article first published online: 22 MAY 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 6, pages 1202–1212, June 2013
How to Cite
An, N., Lin, Y.-W., Mahajan, S., Kellner, J. N., Wang, Y., Li, Z., Kraft, A. S. and Kang, Y. (2013), Pim1 Serine/Threonine Kinase Regulates the Number and Functions of Murine Hematopoietic Stem Cells. STEM CELLS, 31: 1202–1212. doi: 10.1002/stem.1369
Author contributions: Y-W.L.: generated the vav-Pim1-Tx mice; N.A.: performed all experiments and designed research and wrote the paper; S.M.: genotyped the mice; J.N.K.: assisted in flow cytometry experiments; Y.W.: provided stromal cells for CAFC experiments; Z.L. and A.S.K.: participated in research design; Y.K.: designed research and wrote the paper. N.A. and Y.W.L. contributed equally to this article.
Disclosure of potential conflicts of interest is found at the end of this article.
first published online in STEM CELLS EXPRESS February 4, 2013.
- Issue published online: 22 MAY 2013
- Article first published online: 22 MAY 2013
- Accepted manuscript online: 14 MAR 2013 12:01AM EST
- Manuscript Accepted: 25 JAN 2013
- Manuscript Received: 24 SEP 2012
- MUSC Hollings Cancer Center Startup Fund
- Hollings Cancer Center ACS IRG
- ASCO Conquer Cancer Foundation Career Development Award
- NIH. Grant Number: 1K08HL 103780-01A1
- NIH. Grant Number: 3P30CA138313-01S3
- National Institutes of Health
Additional Supporting Information may be found in the online version of this article.
|sc-12-0892_sm_SupplFigure1.tif||663K||Supplementary Figure 1: Generation of Pim1-Tx mice bearing human PIM1 driven by vav- hematopoietic regulatory element and SV40 sequence. (A) The construct used for pronuclear injection. The entire coding DNA sequence of human PIM1 was cloned into vav-regulatory elements and SV40 sequences. (B) Human PIM1 mRNA expression. Human PIM1 mRNA expression was expressed in all hematopoietic tissue but not in non-hematopoietic tissue. Spleen, thymus, bone marrow, and kidney tissues were harvested from WT control mice and Pim1-Tx mice. Messenger RNA was prepared and qRT-PCR performed. The samples were then run on 2% agarose gel. (C) Pim kinase protein expression. Pim1 was over-expressed in the splenocytes of Pim1-Tx mice. Cell lysates were prepared from the splenocytes of Pim1-Tx and WT littermate and were subject to immunoblot analysis using 19F7 Pim1 antibody, which recognizes both human- and mouse-origin of Pim1.|
|sc-12-0892_sm_SupplFigure2.tif||622K||Supplementary Figure 2: Pim1-Tx mice have splenomegaly and increased number of HSPCs in the spleen. (A) Increased spleen weight and size in Pim1-Tx mice. Pim1-Tx mice and WT littermates (1.5 yrs old) were sacrificed and spleen was harvested and weighed. Left panel: three representative mice per group were shown (scale bar indicates 1cm). Right panel: average spleen weight in Pim1-Tx mice and WT littermates (n=11, *p<0.05). (B) Increased LSK HSPCs in spleen in Pim1-Tx mice. Red-blood cell- depleted splenocytes from Pim1-Tx mice and WT littermates were stained with Lineage-specific, Scal-1, and c-Kit antibodies and analyzed on flow cytometry. Total number of LSK HSPCs (calculated by total BM mononuclear cells × %LSK) was shown (n=12, *p < 0.05).|
|sc-12-0892_sm_SupplFigure3.tif||1593K||Supplementary Figure 3: Pim1-Tx mice show no differences in blood cell subsets and have no evidences of myelodysplastic/myeloproliferative disorder. (A) Peripheral blood cell subset analysis. Peripheral blood cells were stained with Gr-1, B220, and CD3 antibody. The absolute numbers of these cell subsets were calculated as described in the Materials and Methods (n=8). (B) Bone marrow morphology. Bone were harvested from Pim1-Tx mice or wild-type control mice, fixed, sectioned, stained with H/E and examined under microscope (representative of 5 mice in each group, scale bar indicates 20μm). Pim1-Tx mice showed no evidences of myelodysplastic/myeloproliferative disorder.|
|sc-12-0892_sm_SupplFigure4.tif||963K||Supplementary Figure 4: Pim1-Tx mice show competitive repopulating units (CRU) comparable to WT littermates at 4 months post transplant. (A) CRUs measured at 6 weeks post transplant. (B) CRUs measured at 4 months post transplant. Various doses of male LSK+CD34- HSCs (15, 45, and 150 cells/recipient mouse) were mixed with 1.5×105 WT female FVB BM cells and transplanted into lethally irradiated female FVB mice. Male donor engraftment was estimated as described at 6 weeks (A: peripheral blood samples; n=29 for WT and n=27 for Pim1-Tx) and 4 months (B: BM samples; n=29 for WT and n=24 for Pim1-Tx) post transplant. Poisson statistical analysis of limitingdilution was performed to allow for estimation of CRUs in each group. The plot shows the percentage of recipient mice containing less than 1% donor male cells at 6 weeks and at 4 months post transplantation versus the number of cells injected per mouse.|
|sc-12-0892_sm_SupplFigure5.tif||854K||Supplementary Figure 5: Pim1−/− mice have significantly reduced competitive repopulating units. Limiting-dilution assay was performed as described using Pim1−/− and WT mice. Panels A-C are blood samples collected at 6 weeks post transplant (A: scatter plot; B: CRU frequencies; C: limiting dilution plot). Panel D is BM samples collected at 4 months post transplant and shown the CRU frequency in Pim1−/− and WT mice.|
|sc-12-0892_sm_SupplFigure6.tif||1824K||Supplementary Figure 6: Gating strategy to analyze BrdU incorporation in LT-HSCs. BM Lin- cells were labeled with fluorescent-labeled antibodies and analyzed by flow cytometry for the longterm HSCs (LT-HSCs, Lin-Sca-1+c-Kit+CD34-CD135-), Short-term HSCs (ST-HSCs, Lin-Sca-1+c- Kit+CD34+CD135-) and multipotent progenitors (MPPs, Lin-Sca-1+c-Kit+CD34+CD135+). BrdU incorporation was analyzed for LT-HSCs.|
|sc-12-0892_sm_SupplFigure7.tif||384K||Supplementary Figure 7: Pim1 regulates LSK cell expansion in vitro. Sorted LSK cells (6,000/well) from WT, Pim1-Tx and Pim1−/− mice were cultured in StemSpan media supplemented with SCF, TPO-1 and Flt3 (100 ng/ml for each) for 4 days. Total cell numbers were counted and averaged from 3-4 wells/group (data are representative of 4 independent experiments).|
|sc-12-0892_sm_SupplFigure8.tif||594K||Supplementary Figure 8: Pim1-Tx LSK cells show a reduced rate of apoptosis. BM Lin- cells were enriched by Lineage cell depletion Kit (Miltenyi Biotec) from WT, Pim1-Tx and Pim1−/− mice. The cells were then stained with biotin-labeled lineage specific antibodies, PE-conjugated Scal-1, and APC-H7-conjugated c-Kit followed by streptavidin-Brilliant-violet-605 and Aqua Live/Dead dye. The cells were then fixed and subsequently stained with PE-Cy7-conjugated Ki-67 antibody and FITC- conjugated Caspase 3 antibody. All the cells were gated on LSK population.|
|sc-12-0892_sm_SupplFigure9.tif||840K||Supplementary Figure 9: Cytokines/growth factors up-regulate the expression of Pim1, but not Pim2 or Pim3. BM Lin- cells isolated from C56B/6 mice were cultured in DMEM medium supplemented with 20% heat-inactive FBS and 100 ng/ml of SCF, IL-3, IL-6, TPO, Flt3 alone or in combination for 24 hrs. Cells were harvested and mRNA isolated for RT-PCR analysis of Pim1 (A), Pim2 (B), and Pim3 (C) gene expression.|
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