WO2007124124A2 - Methods and systems useful in culturing hematopoietic stem cells - Google Patents

Abstract

The present invention relates to methods and compositions for use with hematopoietic stem cell populations in vivo and in vitro, whereby mutant hematopoietic stem cells having mutant PTEN genes can be formed. Systems, tools, and models are provided which are useful in controlling and examining proliferation and differentiation of these populations.

Description

METHODS AND SYSTEMS USEFUL IN CULTURING HEMATOPOIETIC STEM

CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional

Application Serial No. 60/745,368, filed on April 21 , 2006 under 35 U.S.C. §119(e). The '368 application is incorporated by reference as if recited in full herein.

FIELD OF THE INVENTION

[0002] The present invention relates to methods and compositions for use with hematopoietic stem cells in vivo and in vitro. Systems, tools, and models are provided which are useful in controlling and examining proliferation and differentiation of these cells.

BACKGROUND OF THE INVENTION

[0003] Hematopoietic stem cells (HSCs) are clonogenic cells, which possess the properties of both self-renewal and multilineage potential giving rise to all types of mature blood cells. HSCs are responsible for hematopoiesis and undergo proliferation and differentiation to produce mature blood cells of various lineages while still maintaining their capacity for self-renewal. The ability to self- renew maintains the HSC population for the lifespan of an animal and also allows HSCs to repopulate the bone marrow of lethally irradiated congenic hosts. [0004] Early HSC development displays a hierarchical arrangement, starting from long-term (LT-) HSCs, which have extensive self-renewal capability, followed by the expansion state, which corresponds to short-term (ST-) HSCs (having limited self-renewal ability) and proliferative multipotent progenitors (MPPs) (having multipotent potential but no self-renewal capability). MPP is also a stage of priming or preparation for differentiation. A MPP differentiates and commits to either become a common lymphoid progenitor (CLP), which gives rise to all the lymphoid lineages, or a common myeloid progenitor (CMP), which produces all the myeloid lineages. During this process, the more primitive population gives rise to a less primitive population of cells, which is unable to give rise to a more primitive population of cells. The intrinsic genetic programs that control these processes including the multipotential, self-renewal, and activation (or transient amplification) of HSCs, and lineage commitment from MPP to CLP or CMP, remain largely unknown.

[0005] To sustain constant generation of blood cells for the lifetime of an individual, HSCs located in bone marrow niches (Zhang, J. et al. Nature 425, 836- 841 , 2003; Calvi, L. M. et al. Nature 425, 841-846, 2003; Kiel, M. J., et al. Ce// 121, 1109-1121, 2005; Arai, F. et al. Ce// 118, 149-161 , 2004) must achieve a balance between quiescence and activation so that immediate demands for hematopoiesis are fulfilled, while long-term stem cell maintenance is also assured. In adults, homeostasis between the quiescent and activated states of stem cells is important to protect HSCs from losing their potential for self-renewal and, at the same time, support ongoing tissue regeneration (Li, L. and Xie, T. Annu. Rev. Cell. Dev. Biol. 21, 605-631 , 2005). Over-activation and expansion of stem cells risks both eventual depletion of the stem cell population and a predisposition to tumorigenesis. Although some factors important for stem cell activation have been identified (Heissig, B. et al. Ce// 109, 625-637, 2002), the molecular events governing the transition between quiescence and activation are poorly understood. [0006] Phosphatase and tensin homolog (PTEN) functions as a negative regulator of the PI3K/Akt pathway, which plays crucial roles in cell proliferation, survival, differentiation, and migration (Stiles, B. et al. Dev. Biol. 273, 175-184, 2004). The PTEN tumor suppressor is commonly mutated in tumors, including those associated with lymphoid neoplasms, which feature deregulated hematopoiesis (Mutter, G. L. Am. J. Pathol. 158, 1895-1898, 2001; Suzuki, a. et al. Immunity 14, 523-534, 2001). PTEN-deficiency has been associated with expansion of neural and embryonic stem cell populations (Groszer, M. et al. Science 294, 2186-2189, 2001; Kimura, T. et al. Development 130, 1691-1700, 2003). But, the role of PTEN in stem cells and tumorigenesis and the recurrence of tumors is not understood.

SUMMARY OF THE INVENTION

[0007] The present invention provides methods and systems for culturing and studying hematopoietic stem cell populations in vivo and in vitro, whereby mutant hematopoietic stem cells having mutant PTEN genes can be formed. The invention utilizes the regulation of PTEN function to control or influence quiescence, activation, proliferation, and differentiation in hematopoietic stem and mature cells, including progenitor cells and differentiated cells. Thus, the invention also relates to a mutant PTEN animal, such as a genetically engineered mouse that can be used as a model for the study of lymphoid neoplasms such as myeloproliferative disorder (MPD) and leukemia.

[0008] The compositions and systems provided comprise a conditional mutant hematopoietic cell containing a PTEN nucleotide sequence flanked by at least two recombination sites. The cell can be contacted with a recombination activator to produce a hematopoietic cell with expression of a nonfunctional PTEN protein. A suitable recombination activator comprises any site-specific recombinase that recognizes recombination sites flanking a nucleotide sequence and catalyzes recombination of the flanked sequence. It is envisioned that the P1 bacteriophage Cre recombinase, which recognizes LoxP sites, may be utilized to activate recombination of a LoxP-flanked PTEN nucleotide sequence within a hematopoietic cell. Other recombination activators may be used and their use depends upon the recombination sites encoded in the mutant PTEN nucleotide sequence. For example, the D6 bacteriophage Dre recombinase, which recognizes Rox sites, and the Saccharomyces cerevisiae FLP recombinase, which recognizes FRT sites, may also be used if the appropriate recombination sites are included in the mutant PTEN nucleotide sequence. [0009] Alternatively, the cell can be contacted with an inducer that activates expression of the recombination activator to produce a hematopoietic cell with expression of a nonfunctional PTEN protein. A suitable inducer comprises any agent that activates expression of a gene via an inducible system. Such an inducer includes, but is not limited to, polyhpolyC (plpC) or other interferon, anti- progesterone, estrogen, tetracycline, or doxycycline. A suitable inducible system upon which these inducers act includes any system that allows independent and exogenous regulation of the expression of a gene of interest. Inducible systems commonly consist of an inducer and an engineered promoter that has regulatory sites activated by the inducer or an inducer-receptor complex. Such inducible systems include steroid-hormone regulated or binary systems such as interferon regulated, progesterone receptor derived, estrogen receptor derived and tetracycline or doxycycline regulated systems. It is envisioned that the interferon regulated inducible system, induced by an interferon or plpC, will be used. A skilled artisan will recognize that any inducible system and its respective inducer may be used and that optimization of the system will likely be necessary to overcome the possibility of undesirable effects.

[0010] Compositions of the invention comprise the hematopoietic cell being selected from in vivo or in vitro cell populations of mammalian, avian, reptilian, insect, fish or amphibian organisms. It is envisioned that the cell will preferably be selected from a mouse cell population, but could also be from a rat, primate, human, zebrafish, Drosophila, or Xenopus cell population. The cell may be derived from tissue such as bone marrow, spleen or peripheral blood and consist of at least the following cell types: hematopoietic stem cell, long-term hematopoietic stem cell, short-term hematopoietic stem cell, common myeloid progenitor cell, common lymphoid progenitor cell, T progenitor cell, thymocyte cell, T lymphocyte cell, B progenitor cell, B lymphocyte cell, granulocyte-monocyte progenitor, eosinophil progenitor, basophil progenitor, erythroid progenitor, megakaryocyte, erythrocyte, granulocyte, monocyte, eosinophil, neutrophil, basophil, macrophage, dendritic cell, platelets, or bone marrow stromal cell. [0011] Compositions of the invention also comprise a hematopoietic cell containing a mutant PTEN nucleotide sequence rendering the resultant protein encoded by the mutant nucleotide sequence substantially nonfunctional. The mutant cell may arise from a frame shift, point substitution, loss of function, knockout deletion, RNAi inhibition or any conventional deletion mutation. A suitable mutation of the PTEN nucleotide sequence will result in substantially eliminating PTEN protein function. It is envisioned that such a mutation will be conditional and allow the normal function of PTEN until it is so desired to have the nonfunctional PTEN protein expressed. [0012] The compositions of the invention include tissue comprising mutant clonal cells located in bone marrow, spleen, or peripheral blood; a population of cells containing a mutant PTEN nucleotide sequence rendering the resulting mutant PTEN protein nonfunctional; unbalanced lineage commitment; and increased activation of at least one stem cell. The population of cells includes at least one stem cell that divides symmetrically or asymmetrically. Further, the population of cells comprises an unbalanced lineage commitment such that there is an increase in a myeloid or T lymphoid lineage cell type and a decrease in a common lymphoid progenitor or B progenitor cell type. The substantially eliminated functionality of PTEN in the tissue or population of cells is accompanied by the constitutive activation of the PI3K-Akt pathway resulting in impaired stem cell maintenance or unbalanced lineage commitment or both. [0013] The invention also relates to a mutant PTEN organism that can be used as a model for the study of lymphoid neoplasms such as myeloproliferative disorder (MPD) and a variety of leukemias. The mutant PTEN organism comprises a mutant hematopoietic cell that has substantially eliminated PTEN functionality due to mutation of the PTEN nucleotide sequence. The hematopoietic cell may be one of the following cell types: hematopoietic stem cell, long-term hematopoietic stem cell (LT-HSC), short-term hematopoietic stem cell (ST-HSC), common myeloid progenitor cell, common lymphoid progenitor cell, T progenitor cell, thymocyte cell, T lymphocyte cell, B progenitor cell, B lymphocyte cell, granulocyte-monocyte progenitor, eosinophil progenitor, basophil progenitor, erythroid progenitor, megakaryocyte, erythrocyte, granulocyte, monocyte, eosinophil, neutrophil, basophil, macrophage, dendritic cell, platelets, or bone marrow stromal cell. The organism is a conditional PTEN mutant that exhibits a loss in PTEN functionality post-recombination. It is envisioned that the organism is mammalian, but may also be an insect, fish, avian, reptilian, or amphibian. Exemplary organisms include, but are not limited to, a mouse, rat, primate, human cell, zebrafish, Drosophila, or Xenopus. It is envisioned that the preferred organism is a Mx1-Cre⁺PTEN^fi(/f* mutant mouse. The Mx1 -Cre⁺PTEN^f*^/1* mutant organism phenotypically exhibits an expansion in activated stem cell number, myeloproliferative disorder, leukemia, unbalanced lineage commitment, or a combination thereof. A mutant mouse with substantially eliminated PTEN functionality witl also phenotypically exhibit an expansion in activated stem cell number, myeloproliferative disorder, leukemia, unbalanced lineage commitment, or a combination thereof.

[0014] The invention provides an in vitro hematopoietic stem cell cultivation system comprising a substrate; a feeder layer of at least one N-cadherin⁺CD45^" osteoblast cell; and an isolated HSC population comprising at least one HSC with substantially eliminated PTEN functionality. It is envisioned that an HSC of the cultivation system contains a conditional PTEN mutant. Thus, PTEN is functional until the cultivation system is contacted with either the appropriate recombination activator or an inducer of the appropriate recombination activator. [0015] Additionally, it is envisioned that PTEN functionality will be substantially eliminated in an HSC by contacting the cultivation system with a PTEN antisense oligonucleotide. The PTEN antisense oligonucleotide will hybridize with PTEN mRNA and inhibit PTEN mRNA translation, thereby substantially eliminating PTEN functionality. A suitable PTEN antisense oligonucleotide includes siRNA, miRNA, single-stranded DNA phosphorothioate antisense, 2'-O alkyl, peptide nucleic acid (PNA), locked nucleic acid (LNA) or Morpholino antisense. Furthermore, the skilled artisan will recognize that there are additional methods by which PTEN functionality can be substantially eliminated including the use of PTEN antibodies. A skilled artison will recognize that an antibody can be designed to attack the PTEN polypeptide. It is envisioned that use of such an antibody may prevent the functioning of the PTEN polypeptide.

[0016] Methods of the invention for producing a post-recombination Mx1-

Oe⁺PTEN¹^^k _org_anj_sm comprise crossing a Mx1-Cre⁺ organism with a PTEN^^ organism to produce Mx1-Cre⁺PTEN^ficΛk progeny; and administering a recombination activator to the progeny such that Cre-mediated (or other recombinase-mediated) conditional recombination results in substantially eliminating PTEN functionality. A suitable organism may be mammal, insect, fish, bird, reptile, or amphibian. Exemplary organisms include a mouse, rat, primate, human cell, zebrafish, Drosophila, or Xenopus.

[0017J In a preferred example, recombination of mutant PTEN may be initiated by the administration of an interferon such as polyhpoIyC. The interferon will initiate the expression of the recombination activator Cre by the Mx1 promoter. The expression of Cre will catalyze the recombination of the PTEN nucleotide sequence flanked by LoxP sites. The post-recombination organism will then phenotypically exhibit an expansion in activated stem cell number, myeloproliferative disorder, leukemia, unbalanced lineage commitment, or combination thereof. Those of skill in the art will recognize that any of the inducers or recombination activators disclosed herein or known in the art may be substituted in the example to achieve the equivalent result of an organism having non-functional PTEN. [0018] The method for making a genetically engineered model for leukemia comprises generating a mutant PTEN offspring; isolating at least one mutant PTEN HSC; transplanting an isolated mutant PTEN HSC into an irradiated host; if appropriate, administering a recombination activator and/or inducer to excise a portion of the PTEN gene such that a post-recombination mutant organism with substantially eliminated PTEN functionality will result. Successful elimination of PTEN functionality may be determined by detecting an increase in populations of proliferative monocytes and granulocytes in peripheral blood or spleen infiltrated by myeloid cells.

Ϊ0019Ϊ It is envisioned that hematopoietic cells may be isolated from a Mx1-

Cre^PTEN¹*"* mouse; however, such cells may be isolated from any organism with nonfunctional PTEN or capable of having nonfunctional PTEN (conditional mutants). Hematopoietic cells can be isolated using any cell separation technique available including fluorescence activated cell sorting (FACS) or magnetic cell sorting. HSCs can be sorted using antibodies to cell surface markers as follows: Lin-Sca1⁺c-Kit⁺. Once sorted, the HSCs can be transplanted into lethally irradiated hosts. A suitable host comprises the same species of organism from which the HSCs were isolated. If the mutant PTEN is conditional, a recombination activator and optionally an inducer should be applied to render PTEN nonfunctional. Development of leukemia can be detected by monitoring the infiltration of myeloid cells in peripheral blood or spleen and by measuring the number of proliferative monocytes and granulocytes in these tissues by using conventional techniques known in the art.

[0020] The method for making an organism model for myeloproliferative disorder comprises generating a mutant PTEN progeny; if appropriate, administering a recombination activator and/or inducer to excise a portion of the PTEN gene to form a post-recombination mutant organism with substantially eliminated PTEN functionality. Successful elimination of PTEN functionality may be detected by an increase in populations of proliferative monocytes and granulocytes in peripheral blood or spleen infiltrated by myeloid cells. It is envisioned that hematopoietic cells will be isolated from a Mx-I-CTe⁺PTEN¹*⁷¹* mouse; however, such cells may be isolated from any organism with nonfunctional PTEN or capable of having nonfunctional PTEN (conditional mutants). Myeloproliferative disorder development can be detected by monitoring the infiltration of myeloid cells in peripheral blood or spleen and by measuring the number of proliferative monocytes and granulocytes in these tissues. [0021] Methods provided by the invention include substantially or completely blocking PTEN functionality to result in specific phenotypes in vivo or in vitro. Blocking PTEN functionality causes progenitor cells to preferentially differentiate into myeloid or T lymphoid lineages as opposed to common lymphoid progenitors or B lymphoid lineages; allows control of hematopoietic cell development; and allows control of activation of quiescent HSCs. A skilled artisan will recognize the advantageous utility of these methods for further study of lineage commitment, and generating or testing therapies for human disease. [0022] The invention also provides a kit for detecting a mutant PTEN nucleic acid sequence in a hematopoietic cell population that comprises a container and at least one nucleic acid sequence probe. A suitable probe will hybridize to a mutant PTEN sequence such that specific binding by the probe is clearly distinguished from the background. Exemplary probes will comprise at least 18 contiguous nucleotides of mutant PTEN nucleic acid sequence. The probe can also be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more contiguous nucleotides of the mutant PTEN gene. The sequence of the probe will be complementary to the mutant PTEN gene. A skilled artisan will recognize that the design of the probe, its size, and the hybridization conditions used will depend on the goals of the artisan.

[0023] The terms "mutant PTEN" and "PTEN mutant" refer to any alteration in the wild type nucleotide sequence of PTEN, inhibition of translation of PTEN, or inhibition of protein presence that results in a nonfunctional protein. Such alterations include, but are not limited to, a frame shift, point substitution, point mutation, deletion, insertion, premature stop codon, or inversion. Alterations also include those rendered by site-specific recombination systems that alter, excise, or invert essential sections of the PTEN nucleotide sequence. PTEN translational inhibition includes and is not limited to RNA interference (RNAi, siRNA, miRNA), DNA antisense, phosphorothioate, 2'-O alkyl, peptide nucleic acid, locked nucleic acid, or Morpholino antisense. Protein inhibition includes, but is not limited to, antibodies designed to attack PTEN polypeptides. A skilled artisan will recognize that there are numerous ways to render PTEN nonfunctional and the technique used will depend on the resources and goals of the artisan.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The application contains at least one drawing executed in color.

Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0025] FIG. 1A shows a PCR analysis of the targeting efficiency of

PolyhPolyC (plpC)-induced inactivation of the PTEN gene in different types of hematopoietic lineage cells, including HSCs. The cells were sorted 5 days post plpC injection. Abbreviations: Pten loxp LSK (pre-recombination HSCs), KO (PTEN knockout), LSK (HSC), LK (progenitor), BM (bone marrow), GM Mad + (myeloid), spleenTer119+ (myeloid cells), B220+ (B lineage cells), CD3+ (early T cells), CD4CD8+ (mature T cells), CD4+ (T cells), CD8+ (T cells), DN (double negative, CD4^"CD8^") and MNC (mononuclear cells).

[0026] FIG. 1B shows a comparison of the number of HSCs (LSK: Lin^"

Sca1⁺cKit⁺) in post-recombination PTEN^Wf*, MXI-CTO⁺PTEN^⁺, and Mx1- Cre⁺PTEN^wfx mice. PTEN™** is essentially a wild type due to the absence of Cre, and Mx1-Cre⁺PTEN^W+ has half the nonfunctional PTEN as Mx1-Cre⁺PTEN^Wfic due to only one allele being floxed, (n= number of mice examined). [0027] FIG. 2A shows the percentage of cells and activation states in bone marrow for HSCs (LSK), LT-HSCs (LSK Flk2-) and ST-HSCs (LSK Flk2+) in control and PTEN-mutant BM 30 days after plpC injection. Asterisk indicates a difference between control and mutant that is significant at the 5% level. FIG. 2B shows the determination of cell cycle status in control and PTEN mutant HSC (LSK) populations by flow cytometric analysis of relative RNA and DNA content assessed by incorporation of pyronin Y and Hoeschst fluorescent dyes, respectively. Quadrants indicate gating used to classify the cells as being in GO, G1, or S+G2/M. Identical staining procedures and gating parameters were used for both control and PTEN mutant cell populations. Compared to control mice, fewer PTEN mutant mouse HSCs are in the GO phase and a higher proportion are in the S+G2/M phase. Not all of the LSK cells in GO are LT-HSCs since other cell types are included in the LSK population. FIG. 2C shows staining with the proliferation marker Ki67 that was used to determine the proportions of cycling (Ki67+) and non-cycling (Ki67-) cells in the HSC (LSK) and progenitor (LK) (LSK and LK identified by c-Kit) populations isolated from control and PTEN mutant BM. [0028] FIG. 3 shows that expression of PTEN in LT-HSCs is subject to cell

cycle regulation. Sorted LSK Flk2 cell (LT-HSC) preparations were stained as follows: with an antibody that recognizes both PTEN and phosphorylated PTEN and counterstained with DAPI to reveal all cell nuclei (FIG. 3A); and with an antibody that recognizes CyclinDI and counterstained with DAPI to reveal all cell nuclei (FIG. 3B); FIG. 3C shows the merge of data represented in FIG. 3A and 3B with PTEN (green), CyclinDI (red), and DAPI (blue). Co-expression between the

+ +

PTEN and the CyclinDI cells appears yellow. FIG. 3D shows co-staining with antibodies that recognize p-PTEN (green) and Ki67 (red), indicating that most p-

+ PTEN cells are Ki67. FIG. 3E shows bone marrow sections from control mice stained with anti-CyclinD1 antibody (green) and DAPI (blue). FIG. 3F shows bone marrow sections from PTEN mutant mice stained with anti-CyclinD1 antibody

+ (green) and DAPI (blue). Note the increased numbers of CyclinDI cells in the

PTEN mutant.

[0029] FIG. 4A shows flow cytometry analysis of HSCs (LSK) on various days (as indicated) after plpC injection in control and PTEN mutant bone marrow (BM). Asterisks indicate a significance of p<0.05. FIG. 4B shows flow cytometry analysis of HSCs (LSK) on various days (as indicated) after plpC injection in control and PTEN mutant peripheral blood (PB). Asterisks indicate a significance of p<0.05. FIG. 4C shows flow cytometry analysis of HSCs (LSK) on various days (as indicated) after plpC injection in control and PTEN mutant spleen. Asterisks indicate a significance of p<0.05.

[0030] FIG. 5A shows a comparison of spleen in vivo colony forming units

(CFU-S) on day 16 post-transplantation of control and PTEN mutant bone marrow cells. FIG. 5B shows a comparison of spleen in vivo colony forming units (CFU-S) on day 16 post-transplantation of control and PTEN mutant spleen cells. FIG. 5C shows a comparison of in vitro colony forming unit assays between control and PTEN mutant bone marrow cells counted on day 12 of culture, (n = number of replicates). FIG. 5D shows a comparison of in vitro colony forming unit assays between control and PTEN mutant peripheral blood cells counted on day 12 of culture, (n = number of replicates). FIG. 5E shows a comparison of in vitro colony forming unit assays between control and PTEN mutant spleen cells counted on day 12 of culture, (n = number of replicates). FIG. 5F depicts the number of colonies formed 3, 6, 9, 12, and 30 days following recombination with plpC treatment between control and PTEN mutant mice. Bone marrow derived cells (BM) plated at 2x10⁴ cells/plate did not exhibit a difference in the number of control and PTEN colonies formed. Peripheral blood derived cells (PB), plated with 0.5ml_ of cell suspension per plate, and spleen derived cells (Sp), plated at 1x10⁵ cells/plate both exhibited a difference in the number of control and PTEN colonies formed.

[0031] FIG. 6A shows an analysis of relative adhesion between control

(gray bar) and PTEN (black bar) mutant cells on fibronectin (FN), Collagen 1 (CoI), and Laminin (Lam). FIG. 6B shows a comparison of the migration rate of hematopoietic cells in the control and PTEN mutant groups. Cell migration was studied using 24-well 6.5mm transwell plates (50μm pore size Coming-Costar Incorporated, New York, NY). Purified Lin- cells (5 * 104) in fully supplemented Iscove's Medium were added to the upper well. Chemotaxis towards 300ng/ml murine SDF-1_(R & D) in the lower chamber was allowed to continue for 3 hours at 37oC/5% CO2 in a humidified atmosphere. Cells were visualized under the microscope and harvested from the lower well for enumeration. [0032] FIG. 7A shows analysis of BM homing ability for control and PTEN

6 mutant BM. 2 x 10 fluorescently labeled BM cells were transplanted into an irradiated host mouse. Graph shows the proportion of labeled cells found to be located in BM eight hours post-transplantation. FIG. 7B shows lodging ability in

7 control and PTEN-mutant BM. 1 * 10 fluorescently labeled BM cells were transplanted into a non-irradiated host mouse. Graph shows the proportion of labeled cells found to be located in BM six and 18 hours post-transplantation. FIG. 7C shows proliferation profiles of BM- and spleen-lodged cells derived from control (blue) and PTEN-mutant (red) BM, assessed 18 hours posttransplantation. Numbers indicate the number of cell divisions the cells in each fluorescence peak have undergone since labeling.

[0033] FIG. 8A illustrates a schematic of a hematopoietic lineage tree. Red arrows indicate the increased population of cells; green arrows indicate decreased population of cells. The blue vertical bar between the pre-pro-and the pro-B lineages indicates a point of developmental blockade. FIG. 8B shows percentage of CLP, CMP, GMP, MEP progenitor cells detected by flow cytometry of control and PTEN mutant BM 30 days after last plpC injection. FIG. 8C shows

+ + + percentage of Gr1 Mad myeloid and B220 B lineage populations detected by flow cytometry of control and PTEN mutant BM 30 days after last plpC injection.

FIG. 8D shows flow cytometry analysis of B lineage development profiling sorted

+ B220 cell populations from control and PTEN mutant mice. Control panel shows the normal position of Pre-Pro-B, Pro-B and mature B cell populations in these profiles. FIG. 8E shows competitive repopulation analysis of control and PTEN

5 mutant BM. Recipient mice were transplanted with 2*10 rescue BM cells together

6 5 with 1 x10 PTEN mutant BM or 2x10 control BM cells. Peripheral blood was collected at indicated time points after transplantation to assess the donor

+ (CD45.2 ) contribution to different lineages. FIG. 8F shows difference in number of donor-derived HSCs in recipient mice 12 weeks after bone marrow transplantation between control and mutant PTEN cells. FIG. 8G shows an analysis of the reconstitution ability of spleen-derived donor cells 2 (blue), 6 (red), and 12 (yellow) weeks post transplantation. Cells stained with FITC-IJn+ markers for HSCs including B220, CD3 and Gr1 Mad were analyzed by flow cytometry. Spleen-derived cells from PTEN mutant animals were able to repopulate the hematopoietic lineages in recipient mice, indicating the existence of HSCs in the PTEN mutant spleen.

[0034] FIG. 9 shows loss of PTEN results in myeloproliferative disorder and leukemia. Occurrence of myeloproliferative disorder in PTEN mutant mice as evidenced by the aberrant behavior and numbers of myeloid cells found in PB (FIGs. 9A and 9B)₁ spleen (FIGs. 9C and 9D) and liver (FIGs. 9E and 9F). All tissues were studied 30 days post plpC injection. Specifically, FIG. 9A shows increased numbers of myelomonocytic cells in the peripheral circulation of PTEN mutant mice. FIG. 9B shows a measure of Gr-1+Mac-1+ cells using flow cytometry in control and PTEN mutant animals. FIG. 9C shows isolated spleens illustrating spenomegaly in PTEN mutant mice. FIG. 9D shows flow cytometry

+ + analyses of Gr1 Mad myeloid cells in spleens of control and PTEN mutants.

FIG. 9E shows isolated livers illustrating hepatomegaly in PTEN mutant mice. FIG. 9F shows myeloid cell infiltration of PTEN mutant liver (left panel) confirmed by Gr1 staining (right panel).

[0035] FIGs. 10 A-F illustrate leukemia formed from the transplantation of

PTEN mutant bone marrow (BM). Flow cytometry analyses of BM from lethally irradiated mice transplanted with control BM (FIGs. 10A and 10D) or PTEN mutant BM (FIGs. 10B, 10C₁ 10E₁ and 10F).

[0036] FIG. 10A shows that mice receiving control BM showed a normal heterogeneity of cell types. Relative heterogeneity of cell types in BM revealed by separating cells according to their light scattering properties (SSC: side scatter; FSC: forward scatter). FIG. 10B shows that mice receiving mutant PTEN BM showed a homogenous cell type reflecting a blast crisis stage of acute myeloid leukemia (AML). Relative heterogeneity of cell types in BM revealed by separating cells according to their light scattering properties (SSC: side scatter; FSC: forward scatter). FlG. 10C shows that that mice receiving mutant PTEN BM showed a homogenous cell type reflecting a blast crisis stage of acute myeloid and lymphoid leukemia (ALL/AML). Relative heterogeneity of cell types in BM revealed by separating cells according to their light scattering properties (SSC: side scatter; FSC: forward scatter). FIG. 10D shows that mice receiving control BM showed a normal heterogeneity of cell types. Relative heterogeneity of cell types in BM revealed by separating cells according to Gr-1 and Mac-1 marker expression. FIG. 10E shows that mice receiving mutant PTEN BM showed a homogenous cell type reflecting a blast crisis stage of acute myeloid leukemia (AML). Relative heterogeneity of eel! types in BM revealed by separating cells according to Gr-1 and Mac-1 marker expression. FIG. 10F shows that that mice receiving mutant PTEN BM showed a homogenous cell type reflecting a blast crisis stage of acute myeloid and lymphoid leukemia (ALL/AML). Relative heterogeneity of cell types in BM revealed by separating cells according to Gr-1 and Mac-1 marker expression.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention demonstrates that mutation of PTEN can result in HSC deregulation and provides a means and system by which to regulate HSCs. Loss of PTEN function results in a reduction in the proportion of HSCs that are quiescent (in GO) and a corresponding increase in the number of actively cycling HSCs. Furthermore, the present invention provides an in vivo model in which to study lymphoid neoplasms including myeloproliferative disease (MPD) and leukemia. Loss of PTEN function predisposes organisms to develop MPD, which transforms into leukemia.

[0038] The present invention relates to methods and compositions for studying hematopoietic stem cell populations in vivo and in vitro, whereby mutant hematopoietic stem cells having mutant PTEN genes can be formed. Regulation of PTEN function helps to control or influence quiescence, activation, proliferation, and differentiation in hematopoietic stem and mature cells, including progenitor cells and differentiated cells. The invention also relates to a mutant PTEN mouse or other transgenic organism that can be used as an animal model for the study of lymphoid neoplasms such as myeloproliferative disorder (MPD) and leukemia. I. Blocking PTEN Functionality

[0039] Blocking PTEN results in the disruption of the regulation of various biochemical signals that regulate homeostasis of HSCs including symmetric and asymmetric division. When PTEN function is blocked, the biochemical pathways are altered, causing increased activation of HSCs, increased proliferation of HSCs₁ and altered differentiation. PTEN function can be blocked by mutagenizing PTEN, expressing PTEN-specific antisense, or using antibodies to attack PTEN. [0040] PTEN function is preferably blocked in vivo by generating a conditional PTEN knockout or by exogenously expressing PTEN-specific antisense, such as silencing RNA oligonucleotides. Due to the embryonic lethality of classical PTEN knockout mice, in which PTEN is nonfunctional from conception, a conditional PTEN knockout organism or animal is preferred. A mutation is made in the wild type PTEN gene or variants of PTEN, such that the nucleotide sequence encodes a loss of functional PTEN polypeptide by altering an essential section of the gene. Importantly, the mutant sequence should be such that it is fully functional throughout the development of the organism until steps are taken to inactivate the nucleotide sequence, rendering the resultant protein nonfunctional. For example, PTEN can be rendered nonfunctional by excising exon 5 as previously described by Lesche, R. et al. Genesis 32, 148-149, 2002 and incorporated herein by reference. Of course, other portions of PTEN may be excised to result in a nonfunctional PTEN using a similar protocol. A conditional PTEN knockout organism can be exogenously regulated such that PTEN functions normally until induced to become nonfunctional. This regulation permits the organism to proceed through development normally and allows the role of PTEN in embryonic development, adult tissue function and tumorigenesis to be studied.

II. Conditional Mutant Organisms

[0041] In order to produce a conditional organism, one of several site- specific recombination systems can be used in combination with one of several inducible promoters. Suitable recombination systems are characterized by allowing PTEN to function normally until recombination is induced to yield a nonfunctional PTEN. Such recombination systems include, but are not limited to the P1 bacteriophage Cre/loxP, the D6 bacteriophage Dre/Rox, and the Saccharomyces cerevisiae FLP/FRT systems. The mechanism by which these recombination systems work is basically the same. For example, the basic Cre- lox reaction is one in which Cre recombinase recognizes a pair of loxP binding sites flanking a genomic segment of interest (floxed). The Cre enzyme brings the two loxP sites, which carry complementary sequence, together. Depending on the orientation of the paired loxP sites, the intervening DNA may be excised or circularized, inverted or translocated. The generation of a floxed PTEN mouse line, in which exon 5 of PTEN is flanked with loxP sequences rendering PTEN nonfunctional when excised, has been described and is incorporated herein by reference (see Lesche, R. et al. Genesis 32, 148-149, 2002). Expression of Cre recombinase can be driven by either a promoter expressed after the critical time in development when PTEN is necessary or an inducible promoter to permit nonfunctional PTEN at any time desired. It is preferred to use an inducible tissue- specific promoter that is expressed in hematopoietic cells including HSCs and progeny thereof. For example, the MX1-Cre mouse line targets Cre expression to HSCs and progeny when induced by polyhpolyC. The generation of the MX1-Cre mouse line has been described and is incorporated herein by reference (see Zhang, J. et al. Nature 425, 836-841, 2003; and Kuhn, R. et al. Science 269, 1427-1429, 1995). Further methods related to generating conditional knockout mice are described in Current Protocols in Molecular Biology, Unit 23, pub. John Wiley & Sons, Inc., 2003 and Current Protocols in Human Genetics, Unit 15, pub. John Wiley & Sons, Inc., 2002 and both are incorporated herein by reference. [0042] In the present invention, MX1-Cre⁺ and PTEN¹*⁷¹* mice progeny are crossed to form a conditional mouse MXI-Oe⁺PTEN**^ (PTEN mutant). This organism can be conditionally mutated after birth to cause deregulated activation of HSCs, unbalanced lineage commitment during differentiation of hematopoietic progenitors, and leukemogenesis. The knockout organism permits conditional excision of a portion of the target PTEN gene upon the injection of a recombination activator into the organism. The knockout animal may be a pre- recombination or post-recombination animal, where the pre-recombination animal is the PTEN mutant animal prior to injection or application of the recombination activator with functional PTEN and the post-recombination animal is the PTEN mutant animal after injection of the activator with nonfunctional PTEN. Once activated and mutated, an inactive PTEN polypeptide is expressed and PTEN regulation of biochemical signals is blocked. III. In Vivo Use of Antisense Oligonucleotides

[0043] Alternatively, PTEN function can be blocked in vivo by exogenously expressing antisense oligonucleotides such as PTEN-specific silencing RNA oligonucleotides (PTEN siRNA). RNA interference provides an alternative approach to knockout mouse models by inducing sequence-specific mRNA degradation with a 21- to 26-nucleotide small interfering RNA, generated by ribonuclease III cleavage of longer double-stranded RNA (ds-RNA). Due to the embryonic lethality of nonfunctional PTEN throughout development, it is also preferred to use an inducible expression system for exogenously regulating expression of PTEN RNAi. While RNA polymerase III (Pol III) gene promoters express long ds-RNA efficiently in mammalian cultured cells, the Pol III promoter is active in all tissues and cannot be used to express long ds-RNA in a tissue- specific manner. Therefore, RNA polymerase Il gene promoters, which are tissue-specific, are preferred as the targeting promoter. One caveat to using RNA Pol Il promoters is the transfer of their gene coding sequences to the cytosol for processing. Long ds-RNA present in the cytosol elicits an interferon-immune response. This problem can be overcome by blocking the transport of the ds-RNA to the cytosol until after it has been processed into to smaller siRNA fragments in the nucleus. Once processed into siRNA and transported to the cytosol, target mRNA is bound and degraded preventing translation of the protein. This method of transgenic knockdown has been successfully used and described by Shinagawa T. and Shunsuke Ishii, Genes & Development 17, 1340-1345, January 2003 and is incorporated herein by reference. Other methods of transgenic knockdown may include using alternative antisense such as DNA, RNA, phosphorothioate, 2'-O alkyl, peptide nucleic acid (PNA), locked nucleic acid (LNA) and Morpholino antisense. Coupling transgenic knockdown technology with an inducible promoter can further expand the usefulness of this in vivo knockdown system. Such inducible systems include steroid-hormone regulated or binary systems such as interferon regulated, progesterone receptor derived, estrogen receptor derived and tetracycline or doxycycline regulated systems (Fussenegger M., Biotechnol. Prog. 17: 1-51, 2001; and Goand H., J. Gene Med. 4: 258-270, 2002, and incorporated herein by reference).

[0044] PTEN function can be blocked in any of a variety of organisms, including a variety of different mammals, insect, fish, or amphibians. Available mammalian organisms include mice, rats, humans, goats, rabbits, guinea pigs, and any of a variety of other mammals. Insects such as Drosophila melanogaster, fish such as zebrafish, and amphibians such as Xenopus can all be used to study the block of PTEN function as well. IV. Culturinq HSCs

[0045] The present invention relates to cell compositions with blocked

PTEN function. A skilled artisan will recognize that cells can be isolated from the above-described animal models in which PTEN is nonfunctional in hematopoietic cells and used for in vitro studies. The isolation and preparation of bone marrow, thymus, spleen, and peripheral blood cells have been described by Arai, R. et al. Cell 118, 149-161, 2004 and incorporated herein by reference. The HSCs can be isolated and treated in vitro with an inducer of the recombination activator to obtain PTEN mutant HSCs and progeny. The conditional mutant HSCs can be studied and used as tools to better understand HSCs and the pathways influencing HSC quiescence, activation, proliferation, and differentiation. [0046] Isolated HSCs can be cultured in vitro using a cultivation system comprising a feeder layer of osteoblastic cells that prevent differentiation of the stem cells. An isolated population of osteoblastic cells characterized by N- cadherin⁺CD45^" can be used for supporting and promoting growth of HSC in vitro. The osteoblast cells can be obtained by flushing bone marrow cells from tibias and femurs or other bones of a selected host into solution. A suitable solution is PBS; however, other solutions may be used, as long as the efficacy of the cells is maintained. Any of a variety of media and solutions can be used to maintain the integrity of the cells. As such, the femurs or tibias will be flushed until a sufficient population is obtained. Additionally, a bone sample from more than one specimen can be flushed to obtain a sufficient cell population. A sufficient amount of cells should be flushed to provide a suitable population for eventual use as feeder cells. Typically, a population equal to at least 1 x 10⁵ cells should be isolated. The population can be determined by using a cell counter. Such an amount is sufficient for at least one starter culture. If larger applications are to be practiced, obviously, a greater number of cells should be isolated. An alternative to flushing the bone sample is to grind the bone. Grinding is often preferred with mouse bones.

[0047] A bone marrow cell sample is obtained and cultured by removing marrow cells from the bone. The population of cells isolated from the bone marrow sample will include hematopoietic and stromal stem cells. As such, the bone marrow will include osteoblasts, mesenchymal, endothelial, fibroblasts, and hematopoietic cells, and isolated stromal and hematopoietic stem cells. The hematopoietic cells will include lymphoid progenitor cells and myeloid progenitor cells. After isolation, the population of bone marrow cells is treated to remove the myeloid, more particularly, the red blood cells. The red blood cells can be lysed using ammonium chloride, for example. The remaining cell types are separated from the lysed red blood cells and are then ready for analysis. Included in the remaining cells are white blood cells, such as lymphocytes, leukocytes, as well as bone marrow cells, such as osteoblasts. [0048] Once the white blood cells are separated from the red blood cells, the osteoblastic cells are separated from the remaining cells. The isolated cells are mixed with cell surface markers enabling separation of the osteoblast cells from the white blood cells and separation into discrete sub-populations. The cell surface markers may be any of a variety of antibodies, which attach to the cell surface of a specific cell type. The antibodies are labeled with any of a variety of labeling compounds. Kits are commercially available, such as FITC-labeled lineage markers, APC-c-Kit, and PE-Sca-1. Other fluorescent cell surface markers may also be used. As is known, the antibody attaches to a specific antigen on the cell surface. In the present case, the fluorescent FITC-labeled antibodies to the cell surface markers N-cadherin (N-cad) and CD45 are mixed with the cells. After a period of incubation, the marked cells are passed through a flow cytometer, such as a FACS sorter, or similar device, whereby individual cells are separated into discrete populations so that the N-cad⁺CD45^" osteoblast cells are separated from the remainder of the osteoblastic cells. It should be noted that there are other ways to separate cells. Importantly, the population of spindle- shaped N-cad⁺CD45^" osteoblastic cells are different from the other osteoblasts, which are bone-matrix forming cells. The isolated N-cad⁺CD45^" osteoblastic cells can be used as niche cells for supporting HSCs. In particular, the osteoblasts can be used as feeder cells to expand stem cell populations in vitro. [0049] The osteoblast cells can be derived from any of a variety of organisms, including a variety of different mammals. Available mammalian osteoblastic cells include mice, rats, humans, goats, rabbits, guinea pigs, and any of a variety of other mammals. [0050] As mentioned, once isolated, the osteoblast cells are used as feeder cells to support HSC growth. The osteoblasts are added to a culture flask, for example, and then washed. Use as feeder cells is initiated by first plating the osteoblasts so that the N-cad⁺CD45^' cells are no longer dividing. Cell division is stopped by irradiating the cells or by treating the cells with mitosis inhibitors such as Mitomycin C. Media is then mixed with the osteoblasts to maintain the viability of the cells. The media can be formed from a variety of constituents, as long as the osteoblast cells are sufficiently supported. The feeder cells are then plated and the PTEN mutant or wild type HSCs are added to the osteoblast feeder cells. V. In Vitro Use of Antisense Oligonucleotides

[0051] Alternatively, PTEN function can be blocked in vitro by the use of antisense oligonucleotides. An isolated PTEN antisense fragment or antisense oligonucleotide that exists intracellular^ can be used to influence HSC proliferation and development, so that the antisense fragment induces HSC activation by inhibiting translation of PTEN polypeptides, which can cause increased activation of HSCs, increased proliferation of HSCs and alteration in differentiation. The antisense fragment can be inserted into the HSC or other hematopoietic cells by methods including, but not limited to, electroporation, transfection, microinjection, micro-vessel transfer, particle bombardment, biolistic particle delivery, and liposome mediated transfer. The isolated PTEN antisense fragment can be synthesized and multiple copies generated in vitro using a sense template, as is known in the art {Current Protocols In Molecular Biology, Unit 1.5, pub, John Wiley & Sons, Inc., 1998 and incorporated herein by reference). Many forms of antisense have been developed and can be broadly categorized into enzyme-dependent antisense or steric blocking antisense. Enzyme-dependent antisense includes forms dependent on RNase H activity to degrade target mRNA, including single-stranded DNA, RNA, and phosphorothioate antisense. For example, an enzyme-dependent antisense includes siRNA/RNAi and methods for use of siRNA/ RNAi are described in Current Protocols in Molecular Biology, Unit 26, pub. John Wiley & Sons, Inc., 2005 and incorporated herein by reference. Steric blocking antisense includes 2'-O alky!, peptide nucleic acid (PNA), locked nucleic acid (LNA) and Morpholino antisense. VL Antibodies and Blocking PTEN Functionality

[0052] Further, PTEN function can be blocked in vivo or in vitro by the use of antibodies. An antibody to a gene product or protein, particularly PTEN, can be used to generate phenotypic changes in a selected host organism. The antibody can be designed to attack the PTEN polypeptide. Use of such an antibody will prevent the functioning of the PTEN polypeptide and, thus, result in increased HSC activation, proliferation, and altered differentiation in vivo or in vitro. An antibody to the wild type or mutant PTEN polypeptide also will be used to detect and monitor the presence of wild type or mutant PTEN in hematopoietic cells. Thus, isolated antibodies, such as anti-PTEN antibody and fragments thereof, where the antibody, acting as an HSC activator, induces HSC proliferation in vitro by inhibiting PTEN function can be used. Anti-PTEN antibodies are made, isolated, and administered to an HSC or hematopoietic cell population in vitro to attack PTEN. Administration of the isolated antibodies to the HSC population may occur by injection, transfection, particle-mediated delivery, liposome encapsulation, diffusion, or micro-vessel encapsulation. Antibodies can be obtained by polyclonal or monoclonal methodologies described in Current Protocols in Immunology, Unit 2, pub. John Wiley & Sons, Inc., 2002 and incorporated herein by reference. VII. Leukemia and MPD Model

[0053] The present invention also relates to myeloproliferative disorder

(MPD) and leukemia mouse models. The PTEN mutant conditional mouse line provides a novel animal model for the investigation of the molecular mechanisms that cause MPD and leukemia in humans. For example, PTEN mutants around day 30 post-recombination exhibited increased populations of proliferative monocytes and granulocytes in peripheral blood and spleen. Furthermore, multiple tissues in these post-recombination PTEN mutants, including liver, were infiltrated by myeloid cells. These are features characteristic of MPD, which in humans will in many cases transform into leukemia at late stages. To generate an animal model of MPD, PTEN functionality needs to be abolished for a substantial amount of time for myeloid cells to infiltrate multiple tissues. To generate a mouse model of leukemia, isolated PTEN mutant HSCs must be transplanted into irradiated wild type mice to overcome the early lethality of nonfunctional PTEN. [0054] Hematopoietic cells can be isolated using any cell separation technique available including fluorescence activated cell sorting (FACS) or magnetic cell sorting. HSCs can be sorted using antibodies to cell surface markers as follows: Lin-Sca1⁺c-Kit⁺. Once sorted, the HSCs can be transplanted into lethally irradiated hosts. A suitable host comprises the same organism from which the HSCs were isolated. If the mutant PTEN is conditional, a recombination activator should be applied to render PTEN nonfunctional. Leukemia development can be detected by monitoring the infiltration of myeloid cells in peripheral blood or spleen and measuring the number of proliferative monocytes and granulocytes in these tissues as described in Kogan, S. C. et al. Blood 100, 238-245, 2003 and incorporated herein by reference.

[0055] The PTEN mutant mouse model and isolated cells can also be used to study cancer stem cells and PTEN-related diseases such as lymphoblastic leukemia/lymphoma, large B-cell lymphoma, anaplastic large cell lymphoma, Cowden syndrome, PTEN hamartoma tumor syndromes, and PTEN hamartoma tumor syndromes. It is envisioned that HSCs can be isolated from the leukemia model after leukemia develops and compared to wild type HSCs to determine characteristics specific to cancer stem cells. Also, HSCs can be isolated at different time points following MPD or leukemia development, from the respective models, and compared to wild type HSCs to compare differences in the two stem cell types throughout disease progression.

[0056] It is envisioned that pinpointing the differences between cancer stem cells and normal HSCs will provide the foundation for targeted cancer therapies that do not adversely affect normal stem cells. For example, such targeted therapies may be based upon, but not limited to, the regulation of b-catenin, Notch, p53, and Notch, WNT, or Sonic Hedgehog signaling pathways. Further, the PTEN mutant mouse model may be used to elucidate the pathways that play a role in the etiology of MPD and leukemia, such as PTEN/AKT/PI3K or BMP and generate molecular biological tools for research and clinical applications for the treatment and diagnosis of MPD and leukemia. VIIL Kits Utilizing PTEN

[0057] The present invention provides utility kits with reagents for identifying the PTEN mutant nucleotide sequence in a hematopoietic cell population in the above-described methods. Accordingly, a PTEN mutant nucleic acid probe, a positive control consisting of PTEN mutant tissue, and a protocol describing their use are provided in the kit, generally comprised within a suitable container.

[00581 The preferred PTEN mutant nucleic acid probe will specifically recognize mutant PTEN and not wild type PTEN. The nucleic acid probe sequence will be derived from the post-recombination sequence generated following exposure to the recombination activator. For example, PTEN rendered nonfunctional by the excision of exon 5 can be identified using a nucleic acid probe that is complementary to the exon 4/exon 6 boundary with the recombination site. A skilled artisan will recognize that the sequence and length of the probe is dependent on the recombination/mutation location and flanking sequence content.

[0059] The kits of the present invention will also typically include a means for containing the reagent containers in close confinement for commercial sale.

Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[0060] Significantly, it is established herein that PTEN function controlled the HSC number by restricting activation and expansion of stem cells, can influence differentiation to specific lineages, and is consistent with data obtained using a Bmpria knockout in which BMP signal is blocked and stem cells are retained in the niche and not activated (Zhang, J. et al. Nature 425, 836-841 ,

2003). PTEN is a downstream effector of BMP activity.

DEFINITIONS

[0061] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology {2nd Ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise. [0062] "Activated mutant" is a post-recombination organism, tissue, or cell wherein the mutant is obtained by injection of a recombination activator into a conditional mutant organism, tissue, or cell to induce a mutation event that results in inactivation of the targeted gene. For example, an activated PTEN mutant organism is a post-recombination organism which resulted from PoIyI :C injection of a conditional PTEN mutant organism to yield a nonfunctional PTEN gene. [0063] An "amino acid (aminocarboxylic acid)" is a component of proteins and peptides. All amino acids contain a central carbon atom to which an amino group, a carboxyl group, and a hydrogen atom are attached. Joining together of amino acids forms polypeptides. Polypeptides are molecules containing up to 1000 amino acids. Proteins are polypeptide polymers containing 50 or more amino acids.

[0064] A "conditional mutant" is a pre-recombination organism, tissue, or cell wherein injection of a recombination activator into the conditional mutant organism, tissue, or cell induces a mutation event that results in inactivation of the targeted gene, resulting in formation of an activated PTEN mutant organism. [0065] A "conditional PTEN mutant knockout organism" can be a pre- recombination or post-recombination PTEN mutant organism. An example of a conditional PTEN mutant knockout organism is a Mx1-Cre⁺PTEN^fx/fic organism.

The mutant organism may be a mouse. Upon administration of a recombination activator, such as PoIyI :PolyC, to the pre-recombination PTEN mutant organism, a post-recombination PTEN mutant organism is formed in which the cells may contain a mutant PTEN nucleic acid sequence. The recombination activator may be administered either prenatally or postnatally to induce PTEN mutation in the cells.

[0066] "Differentiation" occurs when a cell transforms itself into another form. For example, a hematopoietic stem cell (HSC) may differentiate into cells of the lymphoid or myeloid pathways. The HSC might differentiate into lymphocytes, monocytes, polymorphonuclear leukocytes, neutrophils, basophils, or eosinophils.

[0067] A "gene" is a hereditary unit that has one or more specific effects upon the phenotype of the organism; and the gene can mutate to various allelic forms. The gene is generally comprised of DNA or RNA.

[0068] "Homolog" or "variant" relates to nucleotide or amino acid sequences which have similar sequences and that function in the same way.

[0069] A "host cell" is a cell that receives a foreign biological molecule, including a genetic construct or antibody, such as a vector containing a gene.

[0070] A "host organism" is an organism that receives a foreign biological molecule, including a genetic construct or antibody, such as a vector containing a gene.

[0071] "Knockout" is an informal term coined for the generation of a mutant organism (generally a mouse) containing a null or inactive allele of a gene under study. Usually the animal is genetically engineered with specified wild type alleles replaced with mutated ones. Knockout also refers to the mutant organism or animal. The knockout process may involve administration of a recombination activator that excises a gene, or portion thereof, to inactivate or "knockout" the gene. The knockout organism containing the excised gene produces a nonfunctional polypeptide.

[0072] A "mutation" is defined as a genotypic or phenotypic variant resulting from a changed or new gene in comparison with the Wt gene. The genotypic mutation may be a frame shift, substitution, loss of function, or deletion mutation, which distinguishes the mutant gene sequence from the Wt gene sequence. [0073] A "mutant" is an organism bearing a mutant gene that expresses itself in the phenotype of the organism. Mutants may possess either a gene mutation that is a change in a nucleic acid sequence in comparison to Wt, or a gene mutation that results from the elimination or excision of a sequence. In addition polypeptides can be expressed from the mutants.

[0074] A "nucleotide sequence" is a nucleotide polymer including genes, gene fragments, oligonucleotides, polynucleotides, and other nucleic acid sequences. "Nucleic acid" refers to the monomeric units from which DNA or RNA polymers are constructed, wherein the unit consists of a purine or pyrimidine base, a pentose, and a phosphoric acid group.

[0075] "Plasmids" are double-stranded, closed DNA molecules ranging in size from 1 to 200 kilobases. Plasmids are used as vectors for transfecting a host with a nucleic acid molecule.

[0076] "PolykpolyC (pl:pC)" is an interferon inducer consisting of a synthetic, mismatched double-stranded RNA. The polymer is made of one strand each of polyinosinic acid and polycytidylic acid. PolyhpolyC is 5'-lnosinic acid homopolymer complexed with 5'-cytidylic acid homopolymer (1 :1). PolyhpolyC's pharmacological action includes antiviral activity.

[00771 A "polypeptide" is an amino acid polymer comprising at least two amino acids.

[0078] A "post-recombination mutant organism" is an organism, a targeted gene, or sections thereof, wherein the targeted gene or section has been excised by recombination. The post-recombination organism is called a "knockout" organism. Administration of a recombination activator, such as PoIyIrPoIyC or interferon, can induce the recombination event resulting in target gene excision. A post-recombination PTEN mutant organism is one in which the PTEN gene has been Inactivated.

[0079] A "pre-recombination PTEN mutant organism" is one that has recombination sites flanking regions of the PTEN gene. The pre-recombination organism generally has recombinase-encoded sites that can be induced to express Cre or FIp recombinase, but remain dormant or unexpressed until cells of the organism are exposed to a recombination activator. Administration of the activator to the pre-recombination PTEN mutant organism under proper conditions can transform it into a post-recombination PTEN mutant organism.

[0080] "Proliferation" occurs when a cell divides and results in progeny cells.

[0081] "Self-renewal" occurs when a cell reproduces an exact replicate of itself, such that the replicate is identical to the original stem cell.

[0082] A "stem cell" is defined as a pluripotent or multipotent cell that has the ability to divide (self-replicate) or differentiate for indefinite periods - often throughout the life of the organism. Under the right conditions, or given optimal regulatory signals, stem cells can differentiate to transform themselves into the many different cell types that make up the organism. Stem cells may be distinguishable from progeny daughter cells by such traits as BrdU retention and physical location/orientation. Multipotential or pluripotential stem cells possess the ability to differentiate into mature cells that have characteristic attributes and specialized functions, such as hair follicle cells, blood cells, heart cells, eye cells, skin cells, or nerve cells.

[0083J A "stem cell population" is a population that possesses at least one stem cell.

[0084] "Wild type" is the most frequently observed phenotype in a population, or the one arbitrarily designated as "normal." Often symbolized by "+" or "Wt." The Wt phenotype is distinguishable from mutant phenotype variations. [0085] A "recombination activator" refers to a site-specific DNA recombinase that recognizes specific sites within a sequence and can catalyze site-specific recombination of DNA between two sites. Recombination activators include the P1 bacteriophage recombinase Cre, the D6 bacteriophage recombinase Dre, and the Saccharomyces cerevisiae recombinase FLP. In order for a recombinase to catalyze recombination, it must recognize two specific sites flanking the sequence to be excised. Cre catalyzes recombination of floxed or LoxP-flanked sequence, Dre catalyzes recombination of Rox-flanked sequence, and FLP catalyzes recombination of FRT-flanked sequence. [0086] An "inducible system" refers to the components necessary to allow time-controlled expression of a gene of interest. Typically an inducible system includes a promoter engineered to contain binding sites for specific substrates including mutated progesterone or estrogen receptors, deoxycycline, tetracycline or interferon. The hormone-based systems also require the expression of a mutated estrogen or progesterone nuclear receptor that recognizes receptor modulators such as tamoxifen or anti-progesterone but not endogenous estrogen or progesterone. Typically, expression of these modified receptors is driven by tissue-specific promoters and they remain inactive until contacted by an inducer. The "inducers" or drugs (tamoxifen, anti-progesterone, interferon, or tetracycline) can be administered exogenously to activate expression of a gene of interest driven by the engineered promoter.

[00871 The term "unbalanced lineage commitment" refers to the change in differentiation of stem cells and their progeny due to genetic alterations. Instead of stem cells and their progeny differentiating into all cell types of a system, they differentiate into only a select few cell types. While the specific mechanism regulating the differentiation of a cell to a specific cell type is largely unknown, genetic alterations (such as nonfunctional PTEN) have been shown to affect the cell type a cell preferentially differentiates to, creating an unbalanced lineage commitment.

[0088] A "quiescent or dormant cell" becomes "activated" when it is triggered to enter into the cell cycle. The term "activated" refers to any cell triggered to enter a state of reproduction or doubling and can include a cell entering the cell cycle, cell division, or mitosis. An activated cell can be a pluripotent cell, a totipotent cell, a unipotent cell, a stem cell, or a progeny of a stem cell.

[0089] An "active" cell is a cell undergoing cell division and can be at any point in the cell cycle. An "active" cell also includes "activated" or "cycling" cells. [0090] The term "cycling" refers to any cell that is in a state of reproduction or doubling. Such a cell includes a cell in the cell cycle, cell division, or mitosis and a cell that is active, dividing, or proliferating. A cycling cell can be a stem cell, a pluripotent cell, a totipotent cell, a unipotent cell, a non-stem cell, a precursor cell, a progenitor cell, a differentiated cell, or a progeny of a stem cell. [0091] The term "substrate" as used herein refers to any poly-L-lysine coated tissue culture plate as is known in the art.

[0092] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of th^e invention.

EXAMPLES EXAMPLE 1. PTEN REGULATES STEM CELL ACTIVATION AND

MOBILIZATION.

Conditional PTEN Mouse Model

[0093] To determine the role PTEN plays in the regulation of HSC activation, PTEN function in adult mouse HSCs was disrupted using an inducible knockout mouse model. A polyhpolyC (plpC) inducible Mx1-Cre recombinase line (Kuhn, R. et al. Science 269, 1427-1429, 1995), which can efficiently mediate gene deletion in HSCs, was crossed with a conditional PTEN mutant (PTEN**"*) mouse line (Lesche, R. et al. Genesis 32, 148-149, 2002). The function of PTEN was disrupted by intraperitoneally injecting MX1-Cre+/ PTEN^ mice (PTEN mutants) with plpC (25μg/g bodyweight) on postnatal days 21, 23, and 25, which activates Cre and disables both alleles of PTEN in HSCs and progeny thereof (FIG. 1A). Mice with one wild type PTEN allele (Mx1-Cre+/ PTEN^W+) were used as controls throughout the study since they were phenotypically equivalent to PTEN mutants (FIG. 1B). Effect of PTEN Loss on HSC Populations

[0094] On day 30 after the plpC injections, PTEN mutant mice had fewer

+ + HSCs than controls, as measured by the percentage of Lin Sca-1 c-Kit (LSK) cells in bone marrow (BM) using flow cytometry (FIG. 2A). LSK is a heterogeneous population that includes long-term HSCs (LT-HSCs), which are capable of contributing to hematopoiesis for months or even a lifetime, and short- term HSCs (ST-HSCs) in which reconstitution ability is limited to several weeks (Christensen, J. L. & Weissmann, I. L., PNAS 98, 14541-14546, 2001). The isolation and preparation of bone marrow, thymus, spleen, and peripheral blood cells, and the method for subsequent flow cytometric assays using a CyAn MLE cytometer (Dako Colorado, Inc) have been previously described and incorporated herein by reference (see, Zhang, J. et al. Nature 425, 836-841 , 2003).

[0095] For HSC analysis, mononuclear BM cells were stained with FlTC-

+ Lin markers (CD8, CD3, B220, IgM, Gr1, Ter-119; or this panel of markers with the addition of Mad , CD4, CD5) and with APC-c-Kit, PE-Sca-1 , and PE-Cy5-Flk2.

+ For progenitors, mononuclear BM cells were stained with FITC-Lin markers,

APC-c-Kit, PE-Sca-1, PE-Cy5-IL7Ra, PECy7-CD16/32(Fcr), and Biotin-CD34 followed by Streptavidin-APC-Cy7. The initial decrease in PTEN mutant LSK was mainly due to a decline in the population of LT-HSCs (LSK Flk2 ), whereas the

+ ST-HSCs (LSK Flk2 ) population was unchanged (Fig. 2A). These data indicated that PTEN is required for LT-HSC maintenance. HSC Activation is Dependent on PTEN

[0096] PTEN controls cell cycle entry and progression through inhibition of

PI3K/Akt activity (Stiles, B. et al. Dev. Biol. 273, 175-184, 2004), therefore cell cycle profiles of the HSCs were examined as determined by RNA versus DNA content in LSK cells. PTEN mutants exhibited a two to three fold decrease in the proportion of HSCs in the GO phase and a concomitant increase in the proportion of HSCs in the S+G2M phases (Fig. 2B). Consistent with this result, Ki67 staining revealed that the proportion of cycling HSCs is aberrantly high in PTEN mutants (Fig. 2C).

[0097] The relationship between PTEN expression and cell cycle status was examined due to the increased proportion of proliferating PTEN mutant HSCs. The activity of PTEN is controlled by recruitment to the plasma membrane. Casein kinase Il phosphorylates PTEN at the C-terminus to produce phospho- PTEN (p-PTEN), which is inhibited from recruitment to the plasma membrane and is therefore prevented from acting as a negative regulator of PI3K/Akt activity (Vazquez, F. et al. J. Biol. Chem. 276, 48627-48630, 2001). In vivo p-PTEN is indicative of cells in which Akt is in an active state, therefore p-PTEN and the non- phosphorylated form (non-p-PTEN) were distinguished by staining with one antibody that recognizes both forms of PTEN (pan-PTEN), and another that recognizes only p-PTEN. A strong association between the pan-PTEN signal and the presence of CyclinDI was observed by performing dual label immunofluorescence on sorted LT-HSCs (FIG. 3A-3C). Flow cytometer-sorted Lin Seal c-Kit (LSK), Lin Seal c-Kit Flk2 (LT-HSC) or Lin Seal c-Kit Flk2 (ST- HSC) cells (5,000 per slide) from wild type C57BL/6 mice were fixed in 4% paraformaldehyde for 15 minutes and placed as a drop on coated Cytoslides (Shandon Inc. PA). After air drying for 1 hour at 65°C, slides were washed three times with phosphate buffered saline (PBS) with 0.05% Tween-20. The slides were then blocked with universal blocking reagent (BioGenex) and stained with primary antibodies followed by fluorescent-conjugated secondary antibodies. The primary antibodies used in this study and their dilutions were: mouse anti-active- yff-catenin (1:50, cat. 05-665, Upstate) plus rabbit anti-mouse pan-PTEN (1:50, cat. 9559), p-PTEN (1:50, cat. 9554) and mouse anti-cyclin-D1 (1 :50, cat. 2926), all from Cell Signaling Technology.

[0098] The pan-PTEN signal was found almost exclusively (98% of the

+ time) in cells that were also CyclinDI positive (CyclinDI ), including cells with low as well as high levels of CyclinDI, but >88% of co-staining cells had high

+ + expression of both markers and most (82%) CyclinDI cells were also pan-PTEN

(FIG. 3A-3C). CyclinDI maintains cells at the G1 phase in preparation of the G1/S phase transition, therefore this data suggested that PTEN is regulated through the cell cycle and is predominately expressed in cells at G1. In contrast,

+ + only about half of the CyclinDI cells were found to be p-PTEN although p-PTEN

+ was rarely present in cycling (Ki67 ) HSCs (FIG. 3D). PTEN deficiency markedly increased with the number of CyclinDI + cells in bone marrow sections (FIG. 3E and 3F)₁ although this deficiency likely reflects the combined impact of the mutation on HSCs and on multiple lineages of their progeny. [0099] Since CyclinDI is a known PI3K-Akt pathway target gene, the impact of PTEN deficiency on CyclinDI expression in the bone marrow was analyzed. A marked increase in the overall number of CyclinDI positive cells on bone marrow sections from PTEN mutants (FIG. 3F) when compared with control sections (FIG. 3E) was observed. However, this change likely reflects the combined impact of the mutation not only in the HSCs but also within multiple lineages of their progeny. PTEN Regulates HSC Mobilization

[0100] HSCs are normally located in bone marrow niches. Since the

PTEN-Akt pathway can regulate cell migration, the lack of PTEN on the retention of HSCs in bone marrow was analyzed. The lack of PTEN affected the retention of HSCs in bone marrow niches. HSC (LSK) numbers were assayed by flow cytometry in bone marrow, peripheral blood (PB), and spleen on days 2, 6, 9, 12 and 30 after completing plpC injections. PTEN-deficiency resulted in a slight increase in the percentage of bone marrow HSCs on day 2 but abnormally low bone marrow HSC numbers from day 6 onwards (FIG. 4A), changes that were accompanied by dramatic increases in the HSC population in PB (FlG. 4B) and spleen (FIG. 4C). The total number of nucleated cells was not significantly changed in bone marrow but it was increased 2-3 fold in PB and 8-11 fold in spleens of the PTEN mutant mice compared to control animals on day 30. Thus, PTEN mutation leads to mobilization of HSCs from bone marrow into peripheral blood and spleen, allowing extramedullary (outside the bone marrow) hematopoiesis.

[0101] To functionally assess HSC and progenitor numbers, both in vivo and in vitro quantitative colony forming unit (CFU) assays were used. Bone marrow and spleen cells isolated from control and PTEN mutant mice 30 days after plpC induction were either injected into irradiated mice to assess CFU in the spleen (CFU-S), a simple measure of HSCs previously described by Lian, Z. et al. Stem Cells 17, 39-44, 1999, and incorporated herein by reference, or were used, along with peripheral blood cells, to measure CFU in culture (CFU-C), a measure of multiple myeloid progenitors. In vitro CFU-C assays detect a mixture of multiple myeloid colonies including CFU-E (erythroid), CFU-GM (granulocyte, macrophage) and CFU-GEMM (granulocyte, erythrocyte, monocyte,

5 macrophage). The CFU-C assay was performed using 2*10 BM cells per 35mm

TM diameter dish and MethoCult Media (Cat. 03434; StemCell Technology, Canada) following the manufacture's instructions. Colonies were photographed

5 and counted on day 12. The CFU-S assay was performed using a total of 1*10 nucleated cells from control and PTEN mutant mouse bone marrow and spleen were transplanted into lethally irradiated recipient mice. The recipient mouse spleens were collected on day 16 after transplantation and fixed in Bouin's buffer. The CFU-S were counted under the microscope and documented by photomicroscopy.

[0102] The spleens of recipient mice examined on day 16 indicated that

PTEN mutant-derived bone marrow contained fewer CFU-S than control bone marrow (FIG. 5A). In contrast, PTEN mutant spleen cells formed CFU-S, while those derived from control mice did not (FIG. 5B). HSC mobilization can result in extramedullary hematopoiesis when bone marrow is stressed. Indeed, PTEN- mutant progenitor cells were present ectopically in PB and spleen. In the assay for myeloid progenitor cells, the number of bone marrow derived CFU-C (counted on day 12) was not significantly affected by the PTEN mutation (FIG. 5C), while the numbers of PB- (FIG. 5D) or spleen-derived (FIG. 5E) CFU-C were substantially increased in the PTEN mutants. These functional assays not only confirmed that HSCs carrying the PTEN mutation are mobilized from bone marrow into spleen, but also demonstrated the aberrant presence of progenitor cells in mutant peripheral blood and spleen (FIG. 5F)₁ supporting that PTEN inactivation results in extramedullar hematopoiesis.

[0103] To assess the mechanism of HSC mobilization in PTEN mutants, the ability of HSCs to adhere, migrate and home to bone marrow was examined. PTEN-deficiency did not affect the ability of HSCs to adhere to fibronectin, collagen, or laminin (FIG. 6A). Observation with transwell filter assays and time- lapse video microscopy showed that PTEN mutant cells exposed to stromal cell- derived factor! (SDF-1) were not significantly altered in migration speed or migrating cell number (FIG. 6B). Consistent with the results of these in vitro assays, flow cytometry analysis of HSCs revealed that PTEN deficiency did not alter the expression of either CXCR4, a SDF-1 receptor known to be important for SDF-1 mediated HSC homing, or cr4/5 integrin, which is important for migration and HSC-extracellular matrix interaction (data not shown). PTEN Mutant HSCs Exhibit Defective Homing Capabilities [0104] Using in vivo homing and lodging assays, in which fluorescently labeled bone marrow was transplanted into wild-type hosts and assessed for the ability to home to bone marrow (primarily a property of HSCs), a differential performance of PTEN mutant cells that depended upon the status of the host hematopoietic system was observed. Bone marrow nucleated cells were obtained from control and PTEN mutant mice and labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA, SE) (Sigma) as per manufacture's 6 instructions. The homing assay involved transplantation of 2 * 10 labeled bone

6 marrow cells per lethally irradiated mouse, while in the lodging assay 10 * 10 bone marrow cells were transplanted into each non-irradiated host. The recipient mice from the homing assay were euthanized for tissue isolation 8 hours posttransplantation, while the lodging assays used endpoints of either 6 or 18 hours. In both assays and at all time points, recipient-derived bone marrow and spleen single cell preparations were evaluated by flow cytometry (CyAn MLE) for donor cells expressing CFDA. All data was analyzed using FlowJo software. [0105] Labeled cells from PTEN mutants and from controls performed equally well in homing to bone marrow in irradiated hosts (a treatment that destroys resident HSCs resulting in vacant niches and elevated SDF-1) (FIG. 7A). In contrast, when transplants were performed into hosts with intact bone marrow (with few vacant niches and baseline SDF1 levels), only half as many mutant as control cells were found to be lodged in bone marrow at 6 hour posttransplantation and this difference was greater still by 18 hours (FIG. 7B). Thus, PTEN HSCs are capable of reaching and residing in bone marrow when conditions are conducive for homing, but they do not perform as well as normal HSCs when there are few vacant niches and competing wild-type host HSCs are present.

[0106] The lodging assay also revealed a strong association between proliferation rate and lodging location that may help explain the deficient lodging of PTEN mutant cells in bone marrow (FIG. 7C). Since fluorescent labeling is diluted upon each cell division, it was possible to determine that the labeled control cells that lodged in bone marrow were predominately quiescent (no cell division) up to the 18-hour time point. In contrast, 45% of the cells that lodged in the spleen underwent one, two, or three cell divisions (blue curves in FIG. 7C) in that same time period. Although performed with whole bone marrow, these results were consistent with a recent finding that HSCs that mobilize to the spleen are more proliferative than HSCs retained in bone marrow (Passegue, E. et al. J. Exp. Med. 202, 1599-15611 , 2005). PTEN mutant cells that lodged in bone marrow were quiescent, similar to controls, whereas those that lodged in spleen were more proliferative than controls as evidenced by the significantly greater proportion (60%, p=0.0042) and more prominent peaks of cells that had undergone one or more divisions (red curves FIG. 7C). If bone marrow lodging ability is linked to cell cycle status the reduced lodging of PTEN mutant cells to bone marrow could reflect their increased likelihood of being in an activated state. Furthermore, the decline in PTEN mutant cells lodging in bone marrow between 6 and 18 hours is due to a greater tendency of these cells to become both activated and mobilized.

EXAMPLE 2. PTEN REGULATES LINEAGE COMMITMENT. Hematopoietic Lineage

[0107] The common hierarchical hematopoietic lineage schematic depicts

LT-HSCs giving rise to ST-HSCs, which in turn branch to common myeloid progenitor (CMP) and common lymphoid progenitors (CLP); CLPs, in turn, give rise to T and B lineages while CMPs give rise to megakaryocyte/erythrocyte progenitors (MEP) and granulocyte/monocyte progenitors (GMP) (FIG. 8A). The impact of PTEN deficiency on the hematopoietic lineage 30 days after the last plpC injection was determined using cell surface markers. The isolation and preparation of bone marrow, thymus, spleen, and peripheral blood cells, and the method for subsequent flow cytometric assays using a CyAn MLE cytometer (Dako Colorado, Inc) have been previously described and incorporated herein by reference (see, Zhang, J. et al. Nature 425, 836-841 , 2003).

PTEN Loss Alters Lineage Balance

[0108] For HSC analysis, mononuclear bone marrow cells were stained

with FITC-Lin markers (CD8, CD3, B220, IgM, Gr1 , TeM 19; or this panel with the addition of Mac1,CD4, CD5) and with APC-c-Kit, PE-Sca-1, and PE-Cy5-Flk2 for HSCs. For lineage analyses, antibodies were mixed into different cocktails for different cells: PE-Cy5B220, PE-IgM, APC-CD19 and Biotiπ-CD43 followed by Streptavidiπ-FITC staining for B cells; PE-Mac1 , APC-GrI, PE-Cy5-Ter119 and FITC-CD41 for myeloid cells; FITC-CD3, APC-CD4 and PE-CD8 for mature T cells; and FITC-CD3, CD4 and CD8, with PE-CD44 and APC-CD25 for early T cells.

[0109] Common lymphoid progenitors numbers were significantly lower in

PTEN mutant bone marrow compared with control bone marrow, whereas the

CMP, GMP and MEP populations remain unchanged (FIG. 8B). PTEN-deficiency

+ + also affected more mature lineages. The number of Gr1 Mad myeloid cells was increased and the number of B lymphocytes (measured by B220) was substantially reduced (FIG. 8C). The change in number of B lymphocytes involved both loss of CLP and a developmental block of cells in the pre-pro B

+ lymphoid (CD19 CD43 ) stage (FIG. 8D). Analogous results were obtained under competitive repopulation assays profiling the PB of transplant recipients. PTEN Regulates Myeloid and Lymphoid Lineage Commitment

[0110] Bone marrow nucleated cells from control and PTEN mutant mice

+ +

(CD45.2 background) were mixed with recipient BM nucleated cells (CD45.1 background) respectively and transplanted into lethally irradiated recipient mice

+ 5

(CD45.1 background). Each recipient mouse received either 2*10 control bone

5 6 marrow nucleated cells plus 2*10 compensation cells or 1x10 PTEN mutant

5 bone marrow nucleated cells plus 2*10 compensation cells. After transplantation, peripheral blood was collected from the recipient mice at the indicated time points to investigate the percentage of donor-derived cells based on CD45.2 expression and different lineage contributions. Three months after transplantation the recipient mice were terminated to examine the donor-derived

HSC numbers by enumerating the LSK cells from the CD45.2 population. Tissues from the recipient mice that developed acute leukemia/lymphoma were collected for further cell surface marker staining (see Example 3).

[0111] In mice that received PTEN mutant bone marrow, the percentage of

+ + +

Gr1 Mad myeloid cells and CD3 T cells gradually increased and the percentage

+ of B220 B cells dramatically decreased when compared to recipients of control bone marrow (FIG. 8E). This data indicated that PTEN regulates lymphoid and myeloid lineage commitment.

PTEN Contributes to HSC Homeostasis

[0112] To determine whether PTEN-deficiency impaired the ability of HSCs to reconstitute hematopoiesis a competitive repopulation assay based on transplantation into lethally irradiated recipient mice was performed. The reconstitution ability of donor bone marrow, taken from either PTEN mutants

6 5

(1 ^χ10 cells) or controls (2*10 cells) and identified by the CD45.2 cell surface marker was compared with that of a known quantity of rescue bone marrow

5 +

(2*10 cells; CD45.1 ). A five-fold excess of PTEN mutant cells over control cells was used because pilot experiments revealed the greatly reduced repopulation

+ capacity of PTEN mutant BM. The contribution (percent CD45.2 ) of donor- derived cells, analyzed in peripheral blood at 2, 6, and 12 weeks posttransplantation, progressively decreased for PTEN mutant donors but not for control donors (FIG. 8E). Analysis of bone marrow at the 12-week time point revealed that, although a five fold excess of PTEN mutant cells was transplanted, the percentage of donor-derived LSK cells in bone marrow was decreased in the PTEN mutant-transplanted group to one-eighth of that of the control-transplanted group (FIG. 8F). Thus, the impaired reconstitution ability of PTEN mutants reflects a change in the HSC compartment. Similar results were obtained from transplanted spleen cells (FIG. 8G), indicating that the spleens of PTEN mutant donor animals contained HSCs that were functionally similar to those found in bone marrow and reinforced the conclusion that HSCs carrying the PTEN mutation were aberrantly mobilized from BM.

EXAMPLE 3. LEUKEMIA MODEL Myeloproliferative Disorder Model

[0113] The loss of PTEN function also results in myeloproliferative disorder

(MPD) (Kogan, S. C. et al. Blood 100, 238-245, 2002), which has the potential to transform into acute leukemia. PTEN mutants around day 30, post plpC injection, exhibited increased populations of proliferative monocytes and granulocytes in PB (FIG. 9A and 9B), spleen (FIG. 9C and 9D) and multiple tissues, including liver (FIG. 9E and 9F). The PTEN mutant tissue was infiltrated by myeloid cells. These are all features characteristic of MPD, which in humans will in many cases transform into leukemia at late stages (Kogan, S. C. et al. Blood 100, 238-245, 2002). In PTEN mutants, MPD leads to death of animals around 30-40 days post plpC injection. This MPD can, however, transform into leukemia, which was determined by employing the competitive bone marrow transplantation system described in Example 2 to extend the time window over which the PTEN mutant hematopoietic cells may be followed in vivo. Leukemia Model

[0114] Mice transplanted with PTEN mutant bone marrow displayed the same features of MPD observed in primary (donor) PTEN mutant mice but also subsequently developed severe leukemia or lymphoma and died within 3-4 months. Flow cytometry was used to identify the forms of leukemia/lymphoma generated by transplanting PTEN mutant bone marrow. Mice that received control BM showed a normal heterogeneity of cell types (FlG. 10A), whereas the profiles obtained from recipients of PTEN mutant bone marrow (FIG. 10B and 10C) were more homogenous, reflecting a blast crisis stage of acute myeloid leukemia (AML; 2 of 7 cases) (FIG. 10B and 10E) or acute myeloid and lymphoid leukemia (AML/ALL: 5 of 7 cases) (FIG. 10C and 10F). Two of the five AML/ALL mjce progressed further and developed T lymphoma. In mice with AML, more than 80% of the bone marrow cells displayed the Gr-1/Mac-1 markers reminiscent of myeloid blast crisis in chronic myeloid leukemia (FIG. 10E). [0115] Mice that developed T lymphoid leukemia exhibited one of two phenotypes. In three of five cases the bone marrow was dominated by

+ + CD3 /CD4 T lymphocytes (> 80 %; FIG. 8G), whereas in the other two cases,

+ over 80% of the bone marrow cells were CD44 /CD25 , indicative of an enriched population of pro-T cells (FlG. 10G), and only 10-20% of cells were myeloid (FIG. 10F). These results suggested that loss of PTEN regulation of HSC activation and lineage commitment leads to leukemia development. CITED DOCUMENTS

[0116] The following documents, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0117] Arai, F. et al. Cell 118, 149-161 , 2004

[0118] Calvi, L. M. et al. Nature 425, 841-846, 2003

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[0128] Hale & Marham, The Harper Collins Dictionary of Biology (1991 )

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[0150] Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.

Claims

What is claimed is:

1. A method for expanding a stem cell population comprising modulating PTEN expression in one or more stem cells of the population by culturing the population in a suitable culture media for a period of time sufficient to expand the number of stem cells without significant differentiation of the stem cell population.

2. The method according to claim 1 , wherein the modulation is selected from the group consisting of inhibition of transcription of a PTEN polynucleotide, inhibition of expression of a PTEN polypeptide, and combinations thereof.

3. The method according to claim 2, wherein inhibition of transcription is induced by a change in the nucleotide sequence of PTEN, which change is selected from the group consisting of a frame shift, a point substitution, a point mutation, a deletion, an insertion, a premature stop codon, an inversion, and combinations thereof.

4. The method according to claim 2, wherein inhibition of expression of a PTEN polypeptide is induced by a molecule selected from the group consisting of RNA interference, siRNA, miRNA, DNA antisense, phosphorothioate, 2'-O alkyl, peptide nucleic acid, locked nucleic acid, and morpholino antisense.

5. The method according to claim 1 , wherein the modulation comprises contacting the culture media with an antibody that binds a PTEN polypeptide.

6. The method according to claim 1 , wherein the stem cell population comprises adult stem cells.

7. The method according to claim 6, wherein the adult stem cells are selected from the group consisting of hematopoietic stem cells (HSCs), neural stem cells, epithelial stem cells, and dermal stem cells.

8. The method according to claim 7, wherein the stem cells are HSCs.

9. A method for propagating hematopoietic stem cells (HSCs) in vitro comprising culturing one or more HSCs in a culture media comprising a PTEN modulator for a period of time sufficient to expand the number of HSCs without significant differentiation of the HSCs.

10. The method according to claim 9, wherein the PTEN modulator causes, in one or more stem cells, inhibition of the transcription of a PTEN polynucleotide, inhibition of the expression of a PTEN polypeptide, or both.

11. The method according to claim 10, wherein inhibition of transcription is induced by a change in the nucleotide sequence of PTEN, which change is selected from the group consisting of a frame shift, a point substitution, a point mutation, a deletion, an insertion, a premature stop codon, an inversion, and combinations thereof.

12. The method according to claim 10, wherein inhibition of expression of a PTEN polypeptide is induced by a molecule selected from the group consisting of RNA interference, siRNA, miRNA, DNA antisense, phosphorothioate, 2'-O alkyl, peptide nucleic acid, locked nucleic acid, and morpholino antisense.

13. The method according to claim 9, wherein the modulator is an antibody that binds a PTEN polypeptide.

14. An hematopoietic stem cell (HSC) made by the method according to any one of claims 1 or 9.

15. A method for modulating a PI3K-Akt signal pathway in a stem ceil comprising contacting a stem cell with an effective amount of a PTEN modulator.

16. The method according to claim 15, wherein the stem cell is an adult stem cell.

17. The method according to claim 16, wherein the adult stem cell is selected from the group consisting of hematopoietic stem cells (HSC), neural stem cells, epithelial stem cells, and dermal stem cells.

18. The method according to claim 17, wherein the stem cell is an HSC.

19. The method according to claim 15, wherein the PTEN modulator causes, in one or more stem cells, inhibition of transcription of a PTEN polynucleotide, inhibition of expression of a PTEN polypeptide, or both.

20. The method according to claim 19, wherein inhibition of transcription is induced by a change in the nucleotide sequence of PTEN, which change is selected from the group consisting of a frame shift, a point substitution, a point mutation, a deletion, an insertion, a premature stop codon, an inversion, and combinations thereof.

21. The method according to claim 19, wherein inhibition of expression of a PTEN polypeptide is induced by a molecule selected from the group consisting of RNA interference, siRNA, miRNA, DNA antisense, phosphorothioate, 2'-O alkyl, peptide nucleic acid, locked nucleic acid, and morpholino antisense.

22. The method according to claim 15, wherein the modulator is an antibody that binds a PTEN polypeptide.

23. An in vitro hematopoietic stem cell (HSC) cultivation system, comprising:

a. a substrate; b. a feeder layer comprising at least one N-cad⁺CD45^' osteoblast cell; and c. an isolated cell population comprising at least one hematopoietic stem cell capable of expression of a substantially nonfunctional PTEN.

24. The stem cell cultivation system according to claim 23, wherein the HSC is a conditional mutant.

25. The stem cell cultivation system according to claim 24, wherein expression of nonfunctional PTEN is induced by a site-specific recombination that alters, excises, or inverts at least one section of the PTEN nucleotide sequence necessary for functionality.

26. The stem cell cultivation system according to claim 24, wherein the HSC is a conditional knockout comprising a Rox-flanked, FRT-flanked, LoxP- flanked orfloxed PTEN nucleotide sequence.

27. The stem cell cultivation system according to claim 24, wherein the PTEN mutant is activated by contacting the isolated cell population with a recombination activator.

28. The stem cell cultivation system according to claim 24, wherein the HSC comprises an inducible system selected from the group consisting of steroid hormone system, binary system, interferon regulated, progesterone receptor derived, estrogen receptor derived, deoxycyline and tetracycline regulated systems.

29. The stem cell cultivation system according to claim 28, wherein the isolated cell population is contacted with an inducer to activate expression of a recombination activator.

30. The stem cell cultivation system according to claim 29, wherein the inducer is selected from the group consisting of polyhpolyC, interferon, anti- progesterone, estrogen, deoxycylcine, and tetracycline.

31. The stem cell cultivation system according to claim 23, wherein translation of a PTEN mRNA is inhibited by a PTEN antisense.

32. The hematopoietic stem cell cultivation system according to claim 31, wherein the PTEN antisense is selected from the group consisting of RNA interference, SiRNA, miRNA, DNA antisense, phosphorothioate, 2'-O alkyl, peptide nucleic acid, locked nucleic acid, and morpholino antisense.

33. A method for making a Mx1^~Cre⁺ PTEN^Wf* knockout non-human organism comprising:

a. crossing a PTEN^Wf* non-human organism with an Mx1^'Cre⁺ organism such that a MxrCre⁺ PTEN**"* progeny is produced; and b. administering a recombination activator to the Mx1^"Cre⁺ PTEN**"* progeny to induce Cre-mediated conditional recombination such that PTEN functionality is substantially eliminated.

34. The method according to claim 33, wherein the Mx1^"Cre⁺ PTEN^Mfic non-human organism is selected from the group consisting of mammalian, avian, reptilian, insect, fish, and amphibian.

35. The method according to claim 33, wherein the Mx1^"Cre⁺ PTEN¹*"* non-human organism is selected from the group consisting of mice, rat, primate, zebrafish, Drosophila, and Xenopus.

36. The method according to claim 33, wherein expression of the recombination activator is induced by administration of polyhpolyC or interferon.

37. The method according to claim 33, wherein the Mx1^"Cre⁺ PTEN¹*"* knockout non-human organism expresses a phenotypic change selected from the group consisting of expanded activated stem cell number, myeloproliferative disorder, leukemia, and unbalanced lineage commitment after Cre-mediated conditional recombination.

38. A method for making a non-human animal model for leukemia comprising:

a. isolating a PTEN mutant hematopoietic stem cell (HSC) population, wherein the functionality of PTEN is substantially eliminated; and

b. transplanting the isolated PTEN mutant HSC population into an irradiated non-human host.

39. The method according to claim 38, wherein the irradiated host is selected from the group consisting of mammalian, avian, reptilian, insect, fish, and amphibian animals.

40. The method according to claim 38, wherein the irradiated host is selected from the group consisting of mouse, rat, primate, zebrafish, Drosophila, and Xenopus.

41. The method according to claim 38, wherein the PTEN mutant HSC population is isolated from an organism selected from the group consisting of mammalian, avian, reptilian, insect, fish, and amphibian.

42. The method according to claim 38, wherein the PTEN mutant HSC population is isolated from an organism selected from the group consisting of mouse, rat, primate, human, zebrafish, Drosophila, and Xenopus.

43. The method according to claim 38, wherein the PTEN mutant HSC population comprises at least one cell harboring a PTEN mutation selected from the group consisting of an alteration in a wild type PTEN nucleotide sequence, translational inhibition of PTEN, and PTEN polypeptide inhibition.

44. The method according to claim 43, wherein the alteration in the wild type PTEN nucleotide sequence is selected from the group consisting of a frame shift, point substitution, point mutation, deletion, insertion, premature stop codon, and inversion.

45. The method according to claim 43, wherein the translational inhibition of PTEN is selected from the group consisting of RNA interference, siRNA, miRNA, DNA antisense, phosphorothioate, 2'-O alkyl, peptide nucleic acid, locked nucleic acid, and morpholino antisense.

46. The method according to claim 43, wherein the PTEN polypeptide inhibition comprises an antibody that binds a PTEN polypeptide.

47. The method according to claim 38, wherein the functionality of PTEN is substantially eliminated by site-specific recombination that alters, excises, or inverts a section of the PTEN nucleotide sequence necessary for functionality, which section comprises at least 8 contiguous nucleotides.

48. The method according to claim 38, wherein the PTEN mutant HSC population is contacted with a recombination activator.

49. The method according to claim 38, wherein at least one HSC in the PTEN mutant HSC population comprises an inducible system selected from the group consisting of steroid-hormone system, binary system, interferon regulated, progesterone receptor derived, estrogen receptor derived, and tetracycline regulated systems.

50. The method according to claim 49, further comprising contacting the PTEN mutant HSC population with an inducer to activate expression of a recombination activator.

51. The method according to claim 50, wherein the inducer is selected from the group consisting of polyhpolyC, interferon, anti-progesterone, estrogen, deoxycycline, and tetracycline, and combinations thereof.

52. The method according to claim 38, wherein at least one HSC in the PTEN mutant HSC population is a conditional knockout comprising a Rox-flanked, FRT-flanked, LoxP-flanked orfloxed PTEN nucleotide sequence.

53. A method for making a non-human animal model for a myeloproliferative disorder comprising:

a. generating a PTEN mutant animal, wherein the functionality of PTEN is substantially eliminated in a population of HSC cells; and

b. selecting an animal from (a) having an increase in a population of proliferative monocytes or granulocytes in peripheral blood or spleen infiltrated by myeloid cells.

54. The method according to claim 53, wherein the PTEN mutant animal is selected from the group consisting of consisting of mammalian, avian, reptilian, insect, fish, and amphibian.

55. The method according to claim 53, wherein the PTEN mutant animal is selected from the group consisting of mouse, rat, primate, zebrafish, Drosophila, and Xenopus.

56. The method according to claim 53, wherein the PTEN mutant animal comprises at least one PTEN mutant cell having a mutation selected from the group consisting of an alteration in a wild type nucleotide sequence of PTEN, translational inhibition of PTEN, and PTEN polypeptide inhibition.

57. The method according to claim 56, wherein the alteration in the wild type PTEN nucleotide sequence is selected from the group consisting essentially of a frame shift, point substitution, point mutation, deletion, insertion, premature stop codon, and inversion.

58. The method according to claim 56, wherein the translational inhibition of PTEN is selected from the group consisting of RNA interference, siRNA, miRNA, DNA antisense, phosphorothioate, 2'-O alkyl, peptide nucleic acid, locked nucleic acid, and morpholino antisense.

59. The method according to claim 56, wherein the PTEN polypeptide inhibition comprises an antibody that binds a PTEN polypeptide.

60. The method according to claim 53, wherein the functionality of PTEN is substantially eliminated by site-specific recombination that alters, excises, or inverts a section of the PTEN nucleotide sequence necessary for functionality, which section comprises at least 8 contiguous nucleotides.

61. The method according to claim 53, wherein the PTEN mutant animal is administered a recombination activator.

62. The method according to claim 53, wherein the population of HSC cells comprises an inducible system selected from the group consisting of steroid- hormone system, binary system, interferon regulated, progesterone receptor derived, estrogen receptor derived, and tetracycline regulated systems.

63. The method according to claim 62, wherein the PTEN mutant animal is administered an inducer to activate expression of a recombination activator.

64. The method according to claim 63, wherein the inducer is selected from the group consisting of polykpolyC, interferon, anti-progesterone, estrogen, deoxycycline, tetracycline, and combinations thereof.

65. The method according to claim 53, wherein at least one HSC in the PTEN mutant animal is a conditional knockout comprising a Rox-flanked, FRT- flanked, LoxP-flanked orfloxed PTEN nucleotide sequence.

66. A kit for detecting a mutant PTEN nucleic acid sequence in a hematopoietic cell population comprising:

a. at least one nucleic acid sequence probe, which hybridizes to a mutant PTEN nucleic acid sequence comprising at least 18 contiguous nucleotides of a mutant PTEN polynucleotide; and

b. a container for the probe.

67. A method for modulating progenitor cells to preferentially differentiate into myeloid or T lymphoid lineages relative to common lymphoid progenitors or B lymphoid lineages comprising substantially blocking PTEN functionality in the progenitor cells.

68. A method for modulating the development of a hematopoietic cell (HSC) comprising substantially blocking PTEN functionality in the HSC.

69. A method for controlling activation of quiescent hematopoietic stem cells (HSCs) comprising substantially blocking PTEN functionality in the HSCs.