The role of Src homology 2 domain containing 5\u27 inositol phosphatase 1 (SHIP) in hematopoietic cells

The principal isoform of Src homology (SH) 2-domain containing 5\u27 inositol phosphatase protein 1 (SHIP) is a 145kDa protein primarily expressed by cells of the hematopoietic compartment. The enzymatic activity of SHIP is responsible for hydrolyzing the 5\u27 phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), and thereby preventing the recruitment of pleckstrin homology domain containing effector proteins. Furthermore, SHIP contains protein-protein interaction domains, such as an SH2 domain, two NPXY and several proline-rich motifs. All of these different domains endow SHIP with the capacity to impact signaling pathways important for proliferation, survival, differentiation and activation. Therefore, we hypothesized that SHIP-deficiency could result in the loss of hematopoietic cell homeostasis and functionTo this verify this hypothesis, we first studied the effect of SHIP ablation on hematopoietic stem cell (HSC) proliferation, survival, function and hom ing. Most interestingly we observed that SHIP impacts HSC homeostasis and their ability to home appropriately to the bone marrow. Then, since SHIP was shown to be activated after engagement of the c-mpl receptor by its ligand, thrombopoietin, we studied the impact of SHIP deletion on the function of megakaryocytes, the major target cell of that cytokine. We found that SHIP is also important for homeostasis of the megakaryocyte compartment. Thirdly, we studied the role of SHIP in natural killer (NK) cells biology. We observed that F4 generation SHIP-/- mice have increased NK cells in their spleen and that these cells exhibit a disrupted receptor repertoire. We verified the hypothesis that SHIP helps shape the receptor repertoire of NK cells, mainly through regulation of cell survival and proliferation. Also included, is a study on the role of a SHIP isoform lacking the SH2-domain, called stem cell-SHIP (s-SHIP) in the biology of embryonic stem (ES) cells. To date, this isoform i s expressed by stem/progenitor cells and not by normal differentiated cells. Due to its specific expression pattern, s-SHIP has the potential to have an important role in stem cell biology

SHIP-deficiency to increase megakaryocyte progenitor production

The invention concerns a method for increasing megakaryocyte and megakaryocyte progenitor numbers in vitro or in vivo by suppressing SH2-containing inositol-5-phosphatase (SHIP) function in megakaryocytes or megakaryocyte progenitors expressing the SHIP gene. SHIP function can be suppressed by administering an interfering RNA, or other SHIP inhibitor, to the megakaryocytes or megakaryocyte progenitors in vitro or in vivo

Inhibition of SHIP to enhance stem cell harvest and transplantation

The instant invention teaches the inhibition of SHIP expression, or function, for the increased efficacy of autologous stem cell transplants. In another embodiment, interference with SHIP function can be used to temporarily expand and mobilize the hematopoietic stem cell compartment to assist with leukapheresis, to promote hematopoietic recovery after myeloablation treatments, to deplete target stem cell clones (such a leukemic clones and other tumor stem cell types), and to deplete, or damage, the repopulating ability of the endogenous hematopoietic stem cell pool in order to allow transplanted hematopoietic stem cells to better home and engraft and to promote in vivo expansion and mobilization of other organ-specific stem cell populations (e.g., mesenchymal, mammary)

SHIP deficiency enhances HSC proliferation and survival but compromises homing and repopulation

The SH2 domain–containing inositol 5′-phosphatase-1 (SHIP) has the potential to modulate multiple signaling pathways downstream of receptors that impact hematopoietic stem cell (HSC) biology. Therefore, we postulated that SHIP might play an important role in HSC homeostasis and function. Consistent with this hypothesis, HSC proliferation and numbers are increased in SHIP(–/–) mice. Despite expansion of the compartment, SHIP(–/–) HSCs exhibit reduced capacity for long-term repopulation. Interestingly, we observe that SHIP(–/–) stem/progenitor cells home inefficiently to bone marrow (BM), and consistent with this finding, have reduced surface levels of both CXCR4 and vascular cell adhesion marker-1 (VCAM-1). These studies demonstrate that SHIP is critical for normal HSC function, homeostasis, and homing

SHIP^−/− MK have lowered CXCR-4 receptor expression.

<p>(A) Histogram representing the level of CXCR-4 receptor expression on SHIP^−/− (black line) and WT (gray line) Lin⁻c-kit⁻CD41⁺ cells in BM. Isotype antibody control indicated in the histogram. (B) Represented is the mean±SEM of the mean fluorescence (MFI) intensity of CXCR-4 expression on SHIP^−/− (−/−) (n = 4) and WT (n = 4) Lin⁻c-kit⁻CD41⁺ in BM. Significance was established using the Mann-Whitney test. <i>p</i> values indicated in each graph.</p

SHIP deficient mice have increased number of functional MKP in hematopoietic organs.

<p>(A) BM, spleen, and PB MKP phenotype in SHIP^−/− (−/−) and SHIP^ΔIP/ΔIP (ΔIP/ΔIP) mice. Shown are representative flow cytometry dot plots of CD41 (<i>x</i>-axis) versus c-Kit (<i>y</i>-axis) after gating on live and Lin⁻ cells. MKP (Lin⁻c-Kit⁺CD41⁺) are represented in the right upper quadrants and MK (Lin⁻c-Kit⁻CD41⁺) are represented in the right lower quadrants. Percentages for each population are indicated on each plot. (B) Represented are the mean±standard error mean (SEM) (n = 4 for experimental groups WT C57Bl6 and SHIP^−/−, while n = 6 for experimental groups WT 129SvJ and SHIP^ΔIP/ΔIP) of the absolute numbers of MKP in BM, spleen and PB of SHIP deficient mice (black columns) compared to respective WT littermates (gray columns). Mice strains specified in the graphs. (C) Represented is the mean±SEM (n = 4 for experimental groups WT C57Bl6 and SHIP^−/−, while n = 6 for experimental groups WT 129SvJ and SHIP^ΔIP/ΔIP) of total body MKP numbers determined as the calculated sum of MKP in one whole spleen plus MKP in one femur ×16.6 (since one femur is estimated to contained 6% the total marrow) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003565#pone.0003565-Helgason1" target="_blank">[3]</a>. (D) Represented is the mean±SEM of CFU-MK colonies in BM and spleen of SHIP^−/− (n = 7) and WT (n = 7) littermates. Statistical analysis was established using the Mann-Whitney test. <i>p</i> values indicated on each graph.</p

SHIP deficient MK precursors preserve endomitotic function.

<p>BM and splenocytes from SHIP^−/− and WT mice were enriched for CD41⁺ on AutoMACS, stained with Lin-panel and CD41-biotin/SA-APC, fixed/permeated and stained with PI in the presence of RNase. (A) Representative density plots of ploidy distribution of megakaryocytic cells. Lin⁻CD41⁺ cells were first selected by gating forward and side scatter by area and width. Propidium iodide (<i>x</i> axis) versus forward side scatter (<i>y</i> axis) density plots were then generated to assess ploidy distribution in BM (top panels) and spleen (bottom panels) of SHIP^−/− and WT mice. (B) Represented is the mean±SEM (n = 4 for each experimental group) percentage of BM (top) and spleen (bottom) Lin⁻CD41⁺ cells in different ploidy stages from 2N to 32 N. Statistical analysis was done using the Mann-Whitney test. <i>p</i> values indicated on each graph.</p

Induction of Pluripotent Stem Cells from Mouse Embryonic Fibroblasts by Oct4 and Klf4 with Small-Molecule Compounds

SummarySomatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However, several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently, it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However, NPCs express Sox2 endogenously, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small-molecule combination, BIX-01294 and BayK8644, that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts, which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors, such as Sox2, and improve reprogramming efficiency