WO2009129288A1 - Hematopoietic stem cells characterized by jam-c expression - Google Patents

Abstract

The present invention concerns bone marrow derived hematopoietic stem cells characterized by JAM-C expression, and their isolation, enrichment, purification and use.

Description

HEMATOPOIETIC STEM CFXLS CHARACTERIZED BY JAM-C EXPRESSION

Field of the Invention

The present invention concerns bone marrow derived hematopoietic stem cells characterized by JAM-C expression, and their isolation, enrichment, purification and use.

Background of the Invention Hematopoietic stem cells

Adult hematopoietic stem cells (HSC) have the capacity to maintain a stem cell pool as well as the progeny of the subsequent lineages throughout life. In order to self-renew and continuously differentiate into all blood cell lineages, HSCs must undergo polarization at branching points of differentiation which requires distinct cell fate decisions in the daughter cells (Ho, A. D. and W. Wagner, Curr Opin Hematol, 2007. 14(4): p. 330-6; Faubert, A., J. Lessard, and G. Sauvageau,

Oncogene, 2004. 23(43): p. 7247-55; Wilson, A. and A. Trumpp, Nat Rev Immunol, 2006. 6(2): p. 93-106. Regulatory mechanisms leading to cellular polarity and subsequent asymmetric cell division are provided by intrinsic cues resulting in divisional asymmetry and by extrinsic cues inducing environmental asymmetry. There is increasing evidence that interactions with the environment are key in maintaining the balance between self-renewal and differentiation. Stem cells reside in a special microenvironment termed the "niche" which provides extrinsic regulatory signals that control intrinsic genetic programs required for HSC function Fuchs, E., T. Tumbar, and G. Guasch, Cell, 2004. 116(6): p. 769-78; Moore, K.A. and LR. Lemischka, Science, 2006. 311(5769): p. 1880-5; Scadden, D.T., Nature, 2006. 441(7097): p. 1075-9. A number of cell surface molecules on HSCs have been shown to regulate the maintenance of HSCs. Amongst others these include bone morphogenic proteins (Zhang, J., et al., Nature, 2003. 425(6960): p. 836-41 ), Ca-sensing receptor (Adams, G. B., et al., Nature, 2006. 439(7076): p. 599- 603, Notch (Stier, S., et al., Blood, 2002. 99(7): p. 2369-78; Calvi, L.M., et al., Nature, 2003. 425(6960): p. 841 -6), Duncan, A. W., et al., Nat Immunol, 2005. 6(3): p. 314-22), α4 (Priestley, G.V., et al., Blood, 2006. 107(7): p. 2959-67), and Tie2 (Arai, F., et al., Cell, 2004. 118(2): p. 149-61 ). In addition, transcription profiling of the most primitive HSCs has identified cell junctions proteins, previously not implicated in stem cell functions, to be differentially expressed (Venezia, T. A., et al., PLoS Biol, 2004. 2(10): p. e301; Akashi, K., et al., Blood, 2003. 101(2): p. 383-9; Forsberg, B.C., et al., PLoS Genet, 2005. 1(3): p. e28), and adhesion and junction complexes have been proposed to trigger molecular signals influencing the balance between self-renewing cell division versus differentiation (Ho, A. D. and W. Wagner, supra). Junctional adhesion molecule-C (JAM-C)

The junctional adhesion molecule JAM-C is a member of a family of adhesion molecules belonging to the Ig superfamily. JAMs consist of two extracellular Ig domains followed by a transmembrane domain and a short cytoplasmic tail containing a PDS95/Dig/ZO-l (PDZ) domain- binding motif. Initially, JAM-C was found to be expressed on endothelial cells (Aurrand-Lions, M., et al., J Biol Chem, 2001. 276(4): p. 2733-41 ; Arrate, M.P., et al., J Biol Chem, 2001. 276(49): p. 45826-32; Aurrand-Lions, M., et al., 2001. 98( 13): p. 3699-707), but more recently expression has been detected on a number of different cells like fibroblast (Morris, A. P., et al., Cell Commun Adhes, 2006. 13(4): p. 233-47), smooth muscle cells (Keiper, T., et al., Faseb J, 2005), and spermatids

(Mirza, M., et al., Exp Cell Res, 2006. 312(6): p. 817-30; Gliki, G., et al., Nature, 2004. 431(7006): p. 320-4). Expression of JAM-C on epithelial cells (Zen, K., et al., MoI Biol Cell, 2004. 15(8): p. 3926- 37), platelets (Santoso, S., et al., J Exp Med, 2002. 196(5): p. 679-91 ) and lymphocytes (Arrate, M. P., et al., supra; Johnson-Leger, C. A., et al., Blood, 2002. 100(7): p. 2479-86) is restricted to human tissue. JAM-C interacts heterotypically via its ectodomain with the integrins otjviβa and α_xβ₂ (Zen et al., supra, Santoso et al., supra), JAM-B (Arrate et al., supra, Liang, T. W., et al., J Immunol, 2002. 168(4): p. 1618-26, Lamagna, C, et al., MoI Biol Cell, 2005. 16( 10): p. 4992-5003, Cunningham, S. A., et al.,J Biol Chem, 2002. 277(31 ): p. 27589-92), and the viral receptor CAR (Mirza, M., et al., supra). It can also interact homotypically with itself (Lamagna, C, et al., MoI Biol Cell, 2005. 16( 10): p. 4992-5003; Santoso, S., et al., J Biol Chem, 2005). Through the c-terminal PDZ-binding motif JAM-C associates with the PDZ domain containing proteins ZO-I , Par-3, Par-6, PATJ, and PICK-I (Gliki et al., supra; Ebnet, K., et al., J Cell Sci, 2003. 116(Pt 19): p. 3879-91 ; Reymond, N., et al., FEBS Lett, 2005. 579( 10): p. 2243-9) and localizes to cell-cell junctions (Aurrand-Lions, M., et al., supra; Morris, A. P., et al., supra; Keiper. T., et al., supra; Orlova, V. V., et al., J Exp Med, 2006. 203(12): p. 2703-14; Chavakis, T., et al., J Biol Chem, 2004. 279(53): p. 55602-8).

The broad expression and variety of counter-receptors suggests that JAM-C regulates heterotypic cell-cell interactions, for example leukocyte/endothelial interactions in the immune system, as well as homotypic cell-cell interactions such as cellular junctions in endothelial and epithelial cells Muller, W. A., Trends Immunol, 2003. 24(6): p. 327-34; Weber, C, L. Fraemohs, and E. Dejana, Nat Rev Immunol, 2007. 7(6): p. 467-77; Chavakis, T., K.T. Preissner, and S. Santoso, Thromb Haemost, 2003. 89( 1 ): p. 13-7; Bazzoni, G., Curr Opin Cell Biol, 2003. 15(5): p. 525-30; Ebnet, K., et al., J Cell Sci, 2004. 117(Pt 1 ): p. 19-29; Vonlaufen, A., et al., J Pathol, 2006. 209(4): p. 540-8).

Antibodies against JAM-C and soluble JAM-C fusion proteins can inhibit leukocyte migration in several in vivo models of inflammation (Zen et al., supra; Johnson-Leger et al., supra; Chavakis et al., supra; Vonlaufen, A., et al., supra; Aurrand-Lions, M., et al., J Immunol, 2005. 174(10): p. 6406-15; Ludwig, R.J., et al., J Invest Dermatol, 2005. 125(5): p. 969-76; Palmer, G., et al., Arthritis Res Ther, 2007. 9(4): p. R65), and they can inhibit leukocyte/platelet interactions (Langer, H. F., et al., Arterioscler Thromb Vase Biol, 2007. 27(6): p. 1463-70; Santoso et al., supra). In addition, it has been proposed that JAM-C is necessary for the formation and maintenance of different cell junctions as it co-localizes at cell-cell contacts with adherence and tight junction proteins (Morris et al., supra; Orlova et al., supra; Mandicourt, G., et al., J Biol Chem, 2007. 282(3): p. 1830-7; Satohisa, S., et al., Exp Cell Res, 2005. 310(1): p. 66-78). Antibodies against JAM-C block neovascularization in models of angiogenesis, a process which requires remodeling of endothelial junctions (Orlova et al., supra; Lamagna, C, et al., Cancer Res, 2005. 65(13): p. 5703- 10).

It has been proposed that JAM-Cs role in the diverse events of leukocyte/endothelial transmigration, angiogenesis, and tight junction formation is to polarize cells (Ebnet et al., 2004, supra). JAM-C directly associates with the cell polarity protein PAR-3 targeting it to tight junctions (Ebnet et al., 2003, supra) as well as the observation that JAM-C mutant mice are infertile due to a defect in spermatid differentiation, which requires polarization of round spermatids (Gliki et al., supra). JAM-C appears to be essential for the assembly of a cell polarity complex containing PAR-6, aPKC, PATJ and the small GTPase Cdc42 ensuring elongation and maturation of spermatids. Since JAM-C is a cell surface protein interacting with two cell polarity complexes, Par3-aPKC-Par6 and CRB-PALSl -PATJ, possibly conveying cues provided by the microenvironment to the stem cell regulating cell fate decisions, we investigated the role of JAM-C in hematopoiesis.

Summary of the Invention

The present invention is based, at least in part, on the experimental finding that JAM-C is expressed on hematopoietic progenitors and that expression levels decrease with loss of self-renewal and increased differentiation. Deletion of Jam-C in mice resulted in increased bone marrrow cellularity caused by an increase in myeloid progenitors and granulocytes. Phenotypic analysis, combined with in vitro and in vivo characterization, provide evidence that JAM-C is key in the differentiation of HSCs into myeloid progenitors.

In one aspect, the invention concerns an isolated bone-marrow (BM) derived hematopoietic stem cell (HSC) population enriched in progenitor cells expressing junctional adhesion molecule-C (JAM-C). In a preferred embodiment, the cells are lineage negative.

In various embodiments, the isolated HSC population comprises at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99%, or at least about 99.5%, or at least about 99.9% of the JAM-C expressing progenitor cells. In another embodiment, the HSC population has increased self-renewal capacity and multi- lineage potential.

In another embodiment, the JAM-C expressing progenitor cells comprise cells capable of reconstituting multiple blood cell lineages. In yet another embodiment, the JAM-C expressing progenitor cells comprise cells capable of reconstituting all blood cell lineages.

In a further embodiment, the JAM-C expressing progenitor cells comprise myeloid progenitor cells.

In another aspect, the invention concerns a composition comprising an HSC population of the present invention, which may be a pharmaceutical composition.

In another aspect, the invention concerns a method of isolating a population of primitive hematopoietic stem cells (HSCs) from bone marrow (BM), comprising separating HSCs expressing junctional adhesion molecule-C (JAM-C) from the BM.

In one embodiment, the HSCs are isolated from a lineage negative (Lin ) fraction of the BM.

In another embodiment, JAM-C is used as a single marker for isolating the HSCs

In yet another embodiment, Sca-1 and c-Kit are used as additional markers for isolating the HSCs.

In a further aspect, the invention concerns a method of treating a hematopoietic cancer comprising introducing into a patient in need an HSC of the present invention.

In a still further aspect, the invention concerns a method of treating cell damage caused by chemotherapy treatment comprising introducing into a patient in need an HSC population according to the present invention.

In a different aspect, the invention concerns a method of treating a blood disorder comprising introducing into a patient in need an HSC population according to the present invention. The blood disorder may, for example, be aplastic anemia or sickle cell anemia.

Brief Description of the Drawings

Table 1. Engraftment capacity of JAM-C^pos bone marrow cells. Recipient mice were considered to be reconstituted by CD45.2' cells if the frequencies of CD45.2^* donor-derived myeloid (M, CDl I bVGr-I ") or lymphoid (L, B220\ or CD3ε⁺) cells detected in the peripheral blood at the indicated times after transplantation were greater than background levels (0.13% CD45.2 , 0.4% B220' , and 0% CD3¹ and CDl l b/Gr- l ⁺ as determined by analysis of untransplanted BL6/SJL mice). M + L indicates multilineage reconstitution Table 2. Genotype of litters of heterozygous breeding pairs.

Table 3. Hematologic parameters in wt and homozygous mice. Peripheral blood samples were collected from wild type (n = 10) and homozygous (n =12) mice at 6 -10 weeks of age or from El 8.5 wild type (n = 5) and homozygous (n = 4) embryos. Data are mean +/- SE. The increase of neutrophils is significant at the p = 0.049 level.

Table 4. Hematopoietic compartments in wt and homozygous mice. Peripheral blood samples (wt n = 3 and ko n - 4), spleens (wt n = 5 and ko n = 6), thymi (n = 4), BM (wt n = 5 and ko n = 6), or FL (wt n = 1 1 and ko n = 12) were collected from wild type and homozygous mice. Animals were 4 -10 weeks of age and embryos at stage E14.5. Cells were stained for the indicated cell surface markers and analyzed by Flow cytometry. Data are mean +/- SE.

Figure 1. Expression of JAM-C on hematopoietic stem cells. (A) BM cells from BL6 mice were stained with anti-lineage cocktail (anti-CD3ε, anti-B220, anti-CDl I b, anti-Gr-1 , anti-Terl 19) and the lineage positive (Lin^pos) and lineage negative (Lin^ne8) populations were analyzed for their JAM-C expression. The number above the bracket indicates the percentage of Lin^ne8 cells expressing JAM-C. (B) JAM-C expression on HSCs in the Lin^neg population defined by expression of Sea- 1 and c-Kit (LSK: Lin^", Sca-1 ⁺, c-Kit^f). (C) BM cells were individually stained with lineage markers (anti- CD3ε, anti-B220, anti-CDl I b, anti-Gr-1 , and anti-Terl 19) and analyzed for their expression of JAM- C. (D) Blood, thymus, and spleen cells were stained for JAM-C expression on LSK cells. (E) Staining of the different stem cell populations contained within the LSK gate with anti-JAM-C: LT- HSC (Lin , Sca-r , c-Kit^l, Thyl . T, Flt-3^"), ST-HSC (Lin , Sca-1 ', c-Kit' , Thyl . T, Flt-3 "), MPP (Lin^", Sca-1 ', c-Kit , Thy 1.1 ^", Flt-3^"). The number above the bracket indicates the percentage of cells expressing high levels of JAM-C. (F) Expression of JAM-C on lymphoid progenitors (CLP: Lin^", Sca-l '^nt, c-Kit^int, II7Rα⁴), and myeloid progenitors (CMP: Lin^", Sca-1^", II7Rα^", c-Kit\ CD34⁺, FcγRII/HI^"; GMP: Lin^", Sca-1 ^", II7Rα^", c-Kit⁺, CD34^T, FcγRII/IlL; MEP: Lin^", Sca-1 ^", II7Rα^", c-Kit⁺, CD34^", FcγRII/III^"), The number above the bracket indicates the percentage of cells expressing JAM- C. Shown are representative histograms with anti-JAM-C staining (black histogram line), rabbit serum IgG (filled grey histogram), or anti-JAM-C together with 10x excess of blocking murine JAM- C-Fc (hatched histogram line).

Figure 2. Colony formation potential of JAM-C expressing bone marrow cells. (A) BM cells were stained with anti-lineage cocktail (anti-CD3ε, anti-B220, anti-CDl Ib, anti-Gr-1, anti- Terl 19), anti-Sea- 1 , anti-c-Kit, and anti-JAM-C. Subsequently the Lin ^" cells were sorted into populations lacking JAM-C expression (JAM-C^"), expressing JAM-C (JAM-C ), or expressing Sca-1 and c-Kit (LSK). Numbers indicate the percentage of cells in each gate after the sort. Shown is a representative FACS sort profile. (B) JAM-ClJn^" cells were gated and analyzed for expression of c- Kit and Sca- 1 , which are differentially expressed on hematopoietic progenitors (shown in the far right panel). Staining with control rabbit serum IgG is shown on the left of the dot plot. Numbers indicate the percentage of cells in each gate. (C) Lin^", JAM-C^", JAM-C", and LSK cells were sorted from BM at a density of one cell/well into 96-well plates containing IMDM containing mSCF, mIL-3, mIL-6, liEPO. After 10 days of culture the number of colonies was determined. Data represents mean number of colonies +/- SEM that grew in a total of 300 wells of four independent experiments. (D) Lin^", JAM-C^", JAM-C', and LSK cells were sorted from BM for analysis of in vitro colony-forming ability. 1000 Lin^" and JAM-C^" cells or 200 JAM-C and LSK cells were plated in methylcellulose containing mSCF, mIL-3, mIL-6, hEPO. After 10 days in colonies were assigned scores for the presence of colonies containing single lineages (black bars) or multiple lineages (dark grey bar). Data represent mean +/- SEM on duplicate plates of four independent experiments. Significances are shown on the graph **p < 0.01 and ***p < 0.001. Figure 3. Reconstitution potential of JAM-C^0* bone marrow cells. (A) A total of 500

JAM-C^", JAM-C⁺, and LSK cells were sorted from BM of C57BL6 donor mice and injected intravenously into lethally irradiated BL6/SJL recipients along with 2 x 10⁵ host-type BM cells for rescue. Peripheral blood was stained with anti-CD45.2 for identification of donor progeny and shown as the frequency of donor-derived cells after transfer. Each line represents the frequency of donor- derived cells in a single mouse. The experiment was repeated three times with an average of 5 animals per group. (B) The presence of donor-derived cells within the different blood cell lineages was determined by gating on B220¹ (B cells), CD3ε⁴ (T cells), or CDl I bVGr-I ^f (granulocytes) prior to the assessment of the frequency of CD45.2' cells, LSK cells closed triangles (A), JAM-C⁺ closed circles (•), and JAM-C^" closed circles with dashed line (•). Shown is the average of all mice of three independent experiments with an average of 5 animals per group that showed reconstitution above background level as determined by analysis of untransplanted B6/SJL mice (> 0.4% B220⁺, and > 0% CD3¹ and CDl l b/Gr-1 '). Significances are shown on the graph **p < 0.01 .

Figure 4. Targeted disruption of the JAM-C gene. (A) Schematic diagram of the JAM-C locus including maps of the wild-type (wt) allele and targeting vector, showing the targeted allele (LacZ/Neo: lacZ and neomycin-resistant gene). Black arrows indicate PCR primers (Pl, P5, and

Neo3a). (B) PCR analysis of genomic tail DNA verifying homologous recombination and presence of neo cassette. PCR product of wild type allele is 347 bp and that of the targeted allele 412 bp. (C) RT- PCR analysis showing JAM-C transcript in testis of wild type, heterozygous, and homozygous mice. Actin is shown as control transcript. (D) Western blot analysis showing expression of JAM-C protein in testis of wild type, heterozygous, and homozygous mice. Actin is shown as loading control. (E) Immunohistochemical staining for JAM-C in testis from wild type and homozygous mice and DAPI stained sections of testicular seminiferous tubules from wild type and homozygous mice, demonstrating spermatid arrest with lack of mature spermatozoa in homozygous semniferous tubules. Original magnification 6Ox. Figure 5. Characterization of mature hematopoietic cell populations in Jam-C^{" "} mice. (A) Total number of mononuclear cells in BM (femur and tibia of both hind legs) of wild type and homozygous mice (n = 7). (B and C) Frequency of hematopoietic lineages in BM of wild type and homozygous mice assessed by flow cytometry with antibodies against Terl 19 (erythroid lineage), Gr-I and CDl Ib (myeloid lineage), and B220 (B-cell lineage). (D) Enumeration of erythroid cells, B-cells, and granulocytes in the BM of wild type and homozygous mice. Shown are representative FACS profiles and the numbers on the plots indicate the frequency of cells in the indicated regions. Bar graphs indicate mean ± SE. Significances are shown on the graph **p < 0.01.

Figure 6. Effects of Jam-C deletion on hematopoietic progenitor numbers and activities. Assessment of the frequency (A and B) and total number (B) of lineage negative cells in the BM of wild type and homozygous (n = 7) mice. (C) Flow cytometric analysis of LT- and ST- HSCs (Lin^", Sea- T, c-Kit⁺Thyl .2^l) and progenitors (MPP: Lin^", Sea- T, c-Kit⁺Thyl .2^", CMP: Lin^", Sca-1^", c-Kit^f; CLP: Lin^", Sca-l '^nt, c-Kit^mt) contained within the Lin^" population. Frequency (D) and total number (E) of stem cells and progenitors in the BM of wild type and homozygous mice. Shown are representative FACS profiles and the numbers on the plots indicate the frequency of cells in the indicated regions. Bar graphs indicate mean ± SE. (E) Colony-forming ability of BM cells derived wild type (n = 5) and homozygous (n = 6) mice cultured in methylcellulose containing mSCF, mIL-3, mIL-6, hEPO. After 10 days in culture colonies were assigned scores for the presence of CFU- GEMM, BFU-E, and CFU-GM. Data represent mean ± SE of duplicate plates. Significances are shown on the graph *p < 0.05. Figure 7. Characterization of hematopoietic lineages and progenitors in E14.5 Fetal

Livers. (A) JAM-C expression on E14.5 fetal liver (FL) derived hematopoietic progenitors of wild type and homozygous embryos measured by flow cytometry (LSA: Lin^", Sca-1 ^f, AA4. T). (B) Total number of cells in E14.5 FL (left plot) and hematopoietic progenitors (LSA cells, right plot) of wild type (n = 12) and homozygous (n = 1 1 ) embryos. (C) Frequencies of the different hematopoietic lineages in E14.5 FL of wild type and homozygous embryos were measured by flow cytometry with antibodies against Gr-I and CDl I b (myeloid lineage) and Terl 19 (erythroid lineage). (D) Total number of myeloid and erythroid cells in E14.5 FL of wild type and homozygous embryos. Shown are representative FACS profiles and the numbers on the plots indicate the frequency of cells in the indicated regions. Bar graphs indicate mean ± SE. (E) Colony-forming ability of E14.5 FL cells derived from wild type (n = 12) and homozygous (n = 1 1 ) embryos cultured in methylcellulose containing mSCF, mIL-3, mIL-6, hEPO. After 10 days in culture colonies were assigned scores for the presence of CFU-GEMM, BFU-E, and CFU-GM. Data represent mean ± SE of duplicate plates. (F) Frequency of CD45.2^f donor derived BM cells 8 weeks after the transfer of 2 x 10⁶ FL cells derived from wild type and homozygous embryos (n = 4 in each group) into lethally irradiated BL6 hosts. Data represent mean ± SE. Figure 8. Human Bone Marrow was stained with the human stem cell marker anti-CD34 and analyzed for their JAM-C expression. Shown is a representative histogram with anti-JAM-C staining (black histogram line), isotype control (filled grey histogram).

Figure 9. Amino acid sequence of human JAM-C (SEQ ID NO: 1 ). Figure 10. Nucleic acid sequence of human JAM-C (SEQ ID NO: T).

Detailed Description of the Invention I. Definitions

The term "tissue" as used herein refers to a group or collection of similar cells and their intercellular matrix that act together in the performance of a particular function. The primary tissues are epithelial, connective (including blood), skeletal, muscular, glandular and nervous.

The term "cell" or "cells" as used herein refers to any cell population of a solid or non-solid tissue, especially a bone marrow cell population.

The term "stem cell" is defined herein to refer to any immature cell that can develop into a more mature cell. The stem cells may be pluripotent, bipotent, or monopotent. Monopotent stem cells are also referred to as progenitor cells. Pluripotent stem cells, bipotent stem cells, and progenitor cells are capable of developing into mature cells either directly, or indirectly through one or more intermediate stem or progenitor cells.

The term "hematopoietic stem cell" ("HSC") as used herein refers to pluripotent stem cells or lymphoid or myeloid (derived from bone marrow) stem cells that, upon exposure to an appropriate cytokine or plurality of cytokines, may either differentiate into a progenitor cell of a lymphoid or myeloid cell lineage or proliferate as a stem cell population without further differentiation having been initiated. "Hematopoietic stem cells" include, but are not limited to, colony-forming cell-blast (CFC-blast), high proliferative potential colony forming cell (HPP-CFC) and colony-forming unit- granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) cells. In adults, the majority of hematopoietic stem cells reside in the bone marrow.

The terms "progenitor" and "progenitor cell" as used herein refer to primitive hematopoietic cells that have differentiated to a developmental stage that, when the cells are further exposed to a cytokine or a group of cytokines, will differentiate further to a hematopoietic cell lineage. "Progenitors" and "progenitor cells" as used herein also include "precursor" cells that are derived from some types of progenitor cells and are the immediate precursor cells of some mature differentiated hematopoietic cells. The terms "progenitor", and "progenitor cell" as used herein include, but are not limited to, granulocyte-macrophage colony-forming cell (GM-CFC), megakaryocyte colony-forming cell (Mk-CFC), burst-forming unit erythroid (BFU-E), B cell colony- forming cell (B-CFC) and T cell colony-forming cell (T-CFC). Precursor cells" include, but are not limited to, colony-forming unit-erythroid (CFU-E), granulocyte colony forming cell (G-CFC), colony-forming cell-basophil (CFC-Bas), colony- form ing cell-eosinophil (CFC-Eo) and macrophage colony-forming cell (M-CFC) cells.

As used herein, the term "adult" in reference to bone marrow, includes any bone marrow isolated postnatally, i.e., from juvenile and adult individuals, as opposed to embryos. The term "adult mammal" refers to all post natal individuals, i.e., both juvenile and fully mature mammals, as opposed to embryos.

The term "cytokine" as used herein refers to any cytokine or growth factor that can induce the differentiation of a hematopoietic stem cell to a hematopoietic progenitor or precursor cell and/or induce the proliferation thereof. Suitable cytokines for this purpose include, but are not limited to, erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), thrombopoietin, stem cell factor, interleukin-1 (IL-I ), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), Flt3L, leukemia inhibitory factor (LIF), insulin-like growth factor (IGF), and insulin.

II. Detailed Description

In one aspect, the present invention provides methods and means for the purification and enrichment of a population of bone marrow (BM)-derived lineage-negative primitive hematopoietic stem cells (Lin^" HSCs).

Stem cells are typically identified by the distribution of antigens on the surface of the cells.

For a detailed discussion of stem cells and known stem cell markers see, Stem Cells: Scientific

Progress and Future Directions, a report prepared by the National Institutes of Health, Office of

Science Policy, June 2001, Appendix E: Stem Cell Markers, which is expressly incorporated herein by reference.

The lineage surface antigens are a group of cell-surface proteins that are markers of mature blood cell lineages. Hitherto described lineage surface antigens include, for example, CD2, CD3,

CD4, CD5, CD8, CDl 1 , CDl Ia, NK 1.1 , I-A, Mac- 1 (CDl l b:CD18), CD 14, CD 16, CD 19, CD24,

CD36, CD38, CD45RA, murine Ly-6G, murine TER- 1 19, CD56, CD64, CD86 (B7.2), CD66b, human leucocyte antigen DR(HLA-DR), Gr-I, Terl l 9, and CD235a (Glycophorin A).

Hematopoietic stem cells that do not express significant levels of any of the lineage surface antigens are commonly referred to as "'lineage negative" (Lin ) hematopoietic stem cells. In the experiments described herein, lineage negative cells were defined only by the expression of mouse CD3e (CD3α chain), mouse CDl Ib (only bone marrow), mouse CD45R/B220, mouse Ly-6G and Ly-6C (Gr-I), mouse TER-1 19/erythroid cells (Ly-76), mouse CD5 (Ly-I) (only fetal liver).

All HSCs are found within the lineage negative (Lin ) fraction of the BM and can be further identified by expression of high levels of other markers. Several subpupulations of lineage negative hematopoietic stem cells have been identified that are enriched for hematopoietic stem cells. These include, for example, Lin^" CD34" cells (Krause DS et al., Blood 84: 691-701 (1994)), Lin^" Sea 'kit^' Thy 1 (low) cells (Okada, S., et al., Blood, 80(12): 3044-5 (1992)), and human CD34⁺CD38^~ cells. Primitive, pluri- or totipotent stem cells capable of self-renewal and of generating committed progenitors of the different myeloid and lymphoid compartments.

JAM-identifies HSCs in the BM having long-term repopulation potential as well as non-self- renewing myeloid potential. Including JAM-C as a marker on lineage negative BM cells yields HSC enrichments that are comparable to previously identified markers (Okada et al., 1992, supra) and transfer of these cells lead to long-term multilineage reconstitution in lethally irradiated mice. The identification of simple combinations of markers that allow reliable identification and purification of the most primitive HSCs are of great interest. Using JAM-C as sole marker to purify HSCs from lineage negative BM cells yields HSC enrichments similar to the previously identified markers Sca-1 and c-Kit (Okada et al., 1992, supra). HSCs may be isolated and purified from primary hematopoietic tissue. Bone marrow cells may be collected by physically, enzymatically or chemically dissociating cells in single cell suspension such that a majority of cells to be further processed are no longer attached to other cells from within the original hematopoietic tissue sample. Cells may be further processed in an appropriate isoosmotic salt solution such as phosphate buffered saline (PBS) or Hank's buffered saline solution (HBSS), which may optionally contain protein, e.g. BSA and/or serum and/or further ingredients, such as buffers to maintain physiological pH. Hematopoietic tissue samples for use according to the present invention include tissue samples that have been pre-sorted for HSCs, for example by sorting for CD34 expression, or expression of any HSC marker, pre-enriching by density elutriation, or by any other technique The present invention is not limited by the purification technique or device that takes advantage of the presence or absence of JAM-C on HSCs. Any method suitable for identifying surface proteins may be employed in the various methods of the present invention. For example, HSCs according to the present invention may be identified using fluorescence activated cell sorting analysis (FACS) which uses antibodies conjugated to fluorochromes to directly or indirectly assess the level of expression of a given surface protein on individual cells within a cell preparation of hematopoietic tissue. The expression of or lack of expression of JAM-C on individual cells within the cell preparation may also be assessed using means other than antibody-antigen interaction or fluorescence detection or FACS. HSCs may be physically separated from other cells within a cellular preparation of hematopoietic tissue using any technique known in the art. Common methods known in the art for the separation of specific cells from within a heterogenous population of cells within a hematopoietic cell preparation include, but are not limited to, the use of flow-cytometry, magnetic separation, antibody or protein coated beads, affinity chromatography, or solid-support affinity separation where cells are retained on a substrate according to their expression or lack of expression of a specific protein or type of protein. The degree of purification may vary, depending on the method used and on the desired purity level. Specific purification and enrichment protocols are described in the Example below.

Uses of hematopoietic stem cells

Hematopoietic stem cells are in clinical use for the treatment of hematopoietic cancers

(leukemias and lymphomas) and to treat the damage caused by aggressive, high-dose chemotherapy for the treatment of non-hematopoietic malignancies (cancers of other organs). Hematopoietic stem cells can also be used to treat genetic and acquired blood disorders, such as, for example, aplastic anemia, sickle cell anemia, and autoimmune diseases.

Transplantation of hematopoietic stem cells, in the form of purified stem cell preparations of as part of bone marrow transplant, can involve the use of autologous or allogeneic transplantation. Hematopoietic progenitor cell preparations can be used for bone marrow transplantation.

Human autologous and allogeneic bone marrow transplantations are currently used as therapies for leukemia, lymphoma, and other life-threatening diseases. The possibility to enriched hematopoietic progenitor cell preparations reduces the volume of bone marrow needed for transplantation.

Bone marrow is often damaged by chemotherapy drugs. As a result, blood cell production is rapidly destroyed during chemotherapy treatment, and chemotherapy must be terminated to allow the hematopoietic system to replenish the blood cell supplies before a patient is subjected to further rounds of chemotherapy. Enriched hematopoietic progenitor cell preparations can also be used to supplement chemotherapy.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Example 1

Identification of hematopoietic stem cells characterized by expression of JAM-C

Materials and Methods Mice

Female C57BL/6 (CD45.2 ) mice were purchased from Charles River Laboratories. The congenic strain Igha B6 Ptprca Bβ.SJL used for transfer experiments was generated by crossing

Bβ.SJL-Ptprca Pepcb/BoyJ with B6.Cg-Igha Thy Ia Gpila/J purchased from Jackson Laboratories.

Jam-C ^{" "} mice were generated by Lexicon Genetics Incorporated (Woodlands, TX) by homologous recombination. Gestational age of heterozygous pregnancies was determined by detection of the vaginal plug as embryonic day EO.5. All animals were housed in microisolators in a specific pathogen free facility. Animal experiments were approved by the Institutional Animal Care and Use Committee of Genentech.

Reagents

JAM-C specific antibodies were generated by Josman, LLC (Napa, California). In brief, rabbits were immunized with recombinant his-tagged murine JAM-C ectodomain. JAM-C reactive antibodies were obtained from the bleeds by affinity purification.

Cell isolation

Bone marrow (BM) cells were obtained from mouse femurs and tibia by flushing the central cavity of with DMEM supplemented with 10% FCS. Blood was collected from anesthetized mice by cardiac puncture and transferred to heparin coated tubes (Sarstedt AG & Co, Nϋmbrecht, Germany). Red blood cells were lyzed in ACK buffer (Biosource Invitrogen, Camarillo, CA). For enrichment of early thymic progenitors samples were depleted of single and double positive thymocytes by anti- CD4 (L3T4) and anti-CD8a (Ly-2) coupled magnetic beads (Miltenyi Biotech GmbH, Auburn, CA), according to manufacturers instruction. Fetal livers (FL) were dissected from E14.5-day-old embryos. Flow cytometry analysis and cell sorting

Purified cells were resuspended in PBS containing 10% FCS and 10% mouse serum. Fc receptors were blocked with a blocking antibody to CD16/CD32 at 0.5μg/million (2.4G2; BD Pharmingen, San Diego, CA) and mouse serum IgG at l μg/million (Caltag Laboratories, Burlingame, CA). For staining of GMP and MEP biotin-labeled anti-CD16/32 at 0.25μg/million was used for blocking. Surface expression of JAM-C was detected by staining with 5μg/ml rabbit anti-murine

JAM-C followed by PE conjugated anti-rabbit F(ab)? secondary reagent (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Lineage negative cells were identified by staining with the biotin- or FITC-conjugated mouse lineage panel (BD Pharmingen, San Diego, CA). For analysis of FL CDl I b was excluded from the lineage mix and replaced by CD5 (53-7.3), CD4 (GKl .5), and CD8-biotin (53-6.7). Additional antibodies used were c-Kit (2B8), Sca-1 (E13-161.7), Flt3

(A2F 10.1 ), Thy 1.2 (53-2.1 ), IL7Rα (A7R34), CD34 (S7), FcγRII/III (2.4G2), C 1 qRp (AA4.1 ), B220 (RA3-6B2), CD3ε (145-2C1 1 ), Gr-I (RB6-8C5), CDl I b (M l/70), Ly-76 (Terl 19), and CD45.2 (104). All biotin or directly conjugated fluorescent antibodies used were purchased from eBioscience (San Diego, CA) or BD Pharmingen (San Diego, CA). Biotin-conjugated antibodies were visualized with either PerCP-streptavidin (BD Pharmingen, San Diego, CA), or Pacific Blue-streptavidin

(Molecular Probes, Eugene, OR). Propidium iodide (Molecular Probes, Eugene, OR) was used at 2.5μg/ml for exclusion of dead cells. Cells were analyzed on a two laser FACSscan (BD, Biosciences, San Jose, CA) upgraded by Cytek Development (Fremont, CA), which includes a 632nm laser with two additional PMTs for APC and APC-Cy7, or on a LSRII (BD, Biosciences, San Jose, CA). Cells were sorted on an Aria (BD, Biosciences, San Jose, CA). Data were analyzed with FlowJo (Tree Star, Ashland, OR). In vitro culture analysis

For cloning efficiency assessment, cells were seeded in round bottom 96 well plates in IMDM (Biosource Invitrogen, Camarillo, CA) supplemented with 15% FCS (Sigma, St. Louis, MO), rhlnsulin (Sigma, St. Louis, MO), hTransferrin (Serologicals Corporation, Billerica, MA), 2mM glutamine (Gibco Invitrogen, Gran Island, NY), penicillin and streptomycin (Gibco Invitrogen, Gran Island, NY), 50μM mereaptoethanol (Gibco Invitrogen, Gran Island, NY), 1% BSA (CalBiochem, La JoIIa, CA), lOng/ml rmII-3, 10ng/ml rmII-6, 50ng/ml rmSCF (PeProTech Inc., Rocky Hill, NJ), 2.5μg/ml hEPO (Research Diagnostics Inc., Concord, MA). Colony growth was assessed after 10 days of culture at 37⁰C and 5% CO₂. Methylcellulose cultures were initiated in 35mm culture dishes with the indicated populations of cells purified by Flow cytometry or with BM and FL derived single cell suspensions. Cells were plated into duplicate dished containing Methociilt GF M3434 (StemCell Technologies, Vancouver, BC). Colonies were scored and phenotyped on an inverted phase microscope after 10 days in culture. Transplantation and engraftment assays

For BM transplant experiments, the indicated number of sorted BM cells from C57BL6 mice together with 2 x 10⁵ host-type BM cells, were injected intravenously into the tail veins of lethally irradiated (1200rad) congenic B6/SJL mice. Peripheral blood was obtained from the tail vein at the indicated time points and analyzed by Flow cytometry. For FL engraftment 1x10⁶ FL cells from E 14.5 Jam-C^"'^" or Jam-C"¹ embryos were injected. After 8 weeks the animals were euthanized and bone marrow was harvested and analyzed for the extend of reconstitution. Donor-derived cells were distinguished from host cells by the expression of different CD45 (Ly5, ptprc) antigens (CD45.1 vs CD45.2).

Genotyping Mice were genotyped by PCR. In brief, genomic DNA was extracted from mouse tail clips or yolk sacs of embryos and used for subsequent PCR with ReadyAMP polymerase mix (Sigma, St. Louis, MO) for 30 cycles: 95°C/60s 60°C/60s. The primers used for wild-type allele amplification were 5'-TCA CAT TCC CCT CGA CAT GGC-3' (pi) (SEQ ID NO: 3) and 5'-ATC TGC CAC GGT CCT TCT AGA G-3' (p2) (SEQ ID NO: 4), which yielded a 347bp product. The primers used for mutant allele amplification were 5'-TCA CAT TCC CCT CGA CAT GGC-3' (pi) (SEQ ID NO: 5) and 5'-GCA GCG CAT CGC CTT CTA TC-3' (p3) (SEQ ID NO: 6), which yielded a 412bp product.

RT-PCR

Total RNA was isolated from testis with Rneasy kit (Quiagen, Valencia, CA) and reverse transcribed using MuLV (Applied Biosystems, Foster City, CA). cDNA was amplified using

Advantage polymerase mix (Clontech, Mountain View, CA) for 37 cycles: 95°C/60s 62°C/60s (JAM- C) or 66°C/60s (actin), 68°C/30s. The following primer sequences were used for JAM-C: 5'-GAA GAT CTT CAC CAT GGC GCT GAG CCG G-3' (SEQ ID NO: 7), 5'-CCA TCG ATG GTC AGA TAA CAA AGG ACG ATT TG-3' (956bp) (SEQ ID NO: 8) and actin: 5'-CCA TGG ATG ACG ATA TCG CTG CGC TGG TCG-3' (SEQ ID NO: 9), 5'-CCT AGA AGC ACT TGC GGT GCA CGA TGG AGG-3' (1 135bp) (SEQ ID NO: 10).

Western Blot

Testis was homogenized in lysis buffer (0.5% Tx-100, NaCl, protease inhibitors) and the lysates separated on 4-10% Tris-glycine gels (Invitrogen, Carlsbad, CA), transferred onto nitrocellulose membranes (Invitrogen, Carlsbad, CA) and immmunoblotted with rabbit anti-JAM-C (I μg/ml) or mouse anti-βactin (Abeam, Cambridge, MA) followed by secondary HRP-labeled anti- rabbit or anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA), and visualized by SuperSignal West Pico chemiluminescent (Pierce, Rockford, IL).

Immunohistochemistry (IHC) and immuunofluorescence (IF)

Staining was performed on 5μm thick frozen sections of mouse testis fixed in acetone. For IHC endogenous peroxidase was quenched by glucose oxidase and biotin was blocked with an avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA). After blocking the sections were incubated with anti-JAM-C at O. l μg/ml followed by biotinylated goat anti-rabbit IgG secondary antibody (Vector Laboratories). Staining was visualized usingVectastain ABC Elite reagents (Vector Laboratories) followed by metal enhanced diaminobenzidine (Pierce, Rockford, IL). Slides were counterstained with Mayer's hematoxylin, dehydrated, and mounted. For IF sections were stained with DAPI (Molecular Probes, Invitrogen) and coverslipped with ProLong Gold (Invitrogen).

Hematology

Blood was analyzed with an automated cell counter (Abbott CellDyn 3700). Pl or El 8.5 bleeds were analyzed with a hematology analyzer from Vet ABC and differential counts were determined by manually scoring of blood smears. White blood cell counts were adjusted to the number of nucleated red blood cells present.

Statistics

Statistical significance of the difference between groups was calculated by one-way ANOVA with a t-Test assuming equal variance using JMP (SAS, Cary, NC). All p values < 0.05 are considered significant, and are indicated in the text.

Results

JAM-C expression on hematopoietic progenitors

Adult hematopoeisis occurs in the bone marrow (BM) where the hematopoietic stem cells (HSC) multiply and subsequently differentiate into all blood cell lineages defined by the cell surface expression of specific lineage markers. All HSCs are found within the lineage negative (Lin ) fraction of the BM and can be further identified by expression of high levels of two other markers, the c-Kit receptor and stem cell Antigen-1 (Sca- 1 ) (Okada et al., 1992, supra; Ikuta, K. and I. L. Weissman, Proc Natl Acad Sci U S A, 1992. 89(4): p. 1502-6). HSCs have also been named LSK cells based on the expression pattern of these markers: Lin^"Sca-l c-K.it \ HSCs differentiate into common lymphoid progenitors (CLP) giving rise to the NK, T and B cell lineages (Kondo, M., LL. Weissman, and K. Akashi, Cell, 1997. 91(5): p. 661-72, or common myeloid progenitors (CMP). CMP differentiate into granulocyte/macrophage progenitors (GMP) and the megacaryocyte/erythroid progenitors (MEP) (Akashi, K., et al., Nature, 2000. 404(6774): p. 193-7) giving rise to granulocytes and monocytes, or megacaryocytes, and erythrocytes, respectively. To address a potential role of JAM-C during hematopoiesis we initially performed flow cytometric staining of HSCs. JAM-C is expressed on ~12% of lineage negative cells but not on lineage positive cells in adult BM (Figure I A). Within the lineage negative population JAM-C is highly expressed on LSK cells (Figure I B). We have observed high levels of JAM-C expression on LSK cells of mice up to 8 months of age (data not shown). Staining of BM with lineage markers showed that JAM-C is not expressed on cells committed to specific lineages (Figure 1 C). These include T cells (CD3⁺), B cells (B220^*), myeloid cells (CDl I b⁺), granulocytes (Gr-I⁺), and erythroid cells (Terl 19"). Although most adult HSCs reside in the BM, they can also be found in the peripheral blood of normal animals. HSC in the blood have the capacity to re-engraft functional BM niches at distant sites (Wright, D.E., et al., Science, 2001 . 294(5548): p. 1933-6). In addition, it has been proposed that LSK cells in the blood have efficient T lineage potential and are able to seed the thymus (Schwarz, B.A. and A. Bhandoola, Nat Immunol, 2004. 5(9): p. 953-60). Thus, we examined the levels of JAM-C expression on HSC in the blood, spleen, as well as the thymus. We have observed that JAM-C expression levels are largely decreased on LSK cells in the blood and spleen and almost absent on LSK cells in the thymus (Figure I D), indicating that high levels of JAM-C expression is specific for HSCs in the BM.

The population of LSK cells in the adult BM is composed of stem cells with different self- renewing potential: long-term repopulating HSC (LT-HSC), short-term repopulating HSC (ST-HSC) as well as non-renewing multi-potent progenitors (MPP) (Adolfsson, J., et al., Immunity, 2001. 15(4): p. 659-69; Christensen, J. L. and LL. Weissman, Proc Natl Acad Sci U S A, 2001. 98(25): p. 14541- 6). We observed the highest level of JAM-C expression on LT-HSC. The expression level gradually decreased as stem cells lost their self-renewal potential and became lineage committed (Figure I E and F). We observed low levels of JAM-C expression on 19% of CMPs in comparison with 1 1 % of CLPs (Figure I F). The percentage decreased further in the more mature GMP and MEP populations to 7% and 9%, respectively. These results indicate that JAM-C expression levels correlate with the self-renewal and differentiation potential of HSCs, being highest on LT-HSC, and that expression is maintained longer among myeloid progenitors. Colony-formation of JAM-C expressing BM derived cells

Although JAM-C expression is highest on LT-HSCs, analysis of the expression of the stem cell markers Sca-1 and c-Kit on sorted JAM-C^* cells showed that the population is composed of stem cells and progenitors at different levels of differentiation. About 46% are LSK cells expressing high levels of c-Kit and Sca-1 (Figure 2A). Another 32% had high levels of c-Kit but low levels of Sca-1 and could therefore be classified as CMPs, 3% had intermediate levels of c-Kit and Sca-1 , which corresponds to CLPs, and 12% did not express either.

To determine if JAM-C expressing cells are HSCs with self-renewal capacity, we sorted Lin^" cells from the BM into four populations: a Lin^" populations including all progenitor cells, a Lin^" /JAM-C^" population depleted of JAM-C expressing progenitors (JAM-C^"), a LinVSca-1 7c-Kit⁺ population enriched in stem cells (LSK), and a Lin^"/JAM-C⁺ population enriched in stem cells and early progenitors (JAM-C ) (Figure 2B). Cells were sorted at a density of one cell/well into 96-well plates. After 10 days in culture the number of wells with colonies was assessed (Figure 2C). The plating efficiency of JAM-C' was 20%, about half of the LSK population, which had a plating efficiency of 50%. This correlates with the FACS analysis showing that only 46% Of JAM-C⁺ cells express high levels of the stem cell markers c-Kit and Sca-1 and correspond to LSK cells with self- renewal potential. Sorting on JAM-C expressing cells within the LlN^" population doubled the plating efficiency (p = 0.002). The number was low in the Lin^" and JAM-C^" populations, reaching plating efficiencies of about 10% and 6%, respectively. This result indicates that the JAM-C^* population is composed of stem cells that have the ability to self-renew as well as progenitors that do not self- renew under the provided conditions.

We next analyzed the in vitro differentiation potential of the sorted populations by comparing their colony- form ing ability in methylcellulose. The generation of colonies with a single-lineage is indicative of a more mature progenitor cell producing one lineage. In contrast, the presence of multi- lineage colonies is indicative of more immature progenitor cells with the capacity to differentiate into multiple lineages. The colonies growing from the Lin^" population were composed of a similar percentage of single-lineage colonies (40%) and multi-lineage colonies (60%), showing that this population comprises similar proportions of immature and mature progenitors. Depleting the Lin^" population from cells expressing JAM-C lead to a decrease in multiple-lineage colonies (30%, p = 0.0003) (Figure 2D), indicating that this population is depleted of more immature progenitors that give rise to multiple-lineage colonies. In contrast, including JAM-C as a marker in the sort increased the frequency of multi-lineage colonies (80%, p = 0.002), suggesting that JAM-C identifies progenitors within the Lin^" population that are more immature and have multi-lineage potential. Both the JAM-C^* and LSK sorted populations give rise to similarly differentiated colonies composed to a large extent of multi-lineage colonies (80%). These data suggest that the JAM-C^* population is enriched in early progenitors with multi-lineage potential and that inclusion of JAM-C in the Lin^" sort leads to an increase in immature progenitors.

Lineage potential of JAM-C expressing bone marrow derived cells

To determine the in vivo lineage potential of the sorted JAM-C⁺ population we compared their repopulation potential with that of the JAM-C^" and LSK populations. Sorted cells from B6 mice were injected intravenously into lethally irradiated B6.SJL hosts along with host-type BM. The repopulation capacity was assessed by the donor cell contribution to myeloid and lymphoid lineages in the peripheral blood measured by flow cytometry. Intravenous transfer of the JAM-C^* cells resulted in a slightly elevated level of chimerism as the transfer of LSK cells, assessed by the percentage of CD45.2 positive cells in the blood (Figure 3A and Tablel ). In contrast, the transfer of the JAM-C^" cells resulted in a low and transient wave of donor derived immune cells. Using JAM-C as a marker on lineage negative BM cells leads to the purification of HSC cells that efficiently reconstitute lethally irradiated mice long-term.

The JAM-C' population gave rise to a sustained production of T cells, B cells and myeloid cells, indicating that like LSK subsets, the JAM-C⁺ population is comprised of HSCs (Figure 3B). In comparison to the LSK population, the JAM-C⁺ cells showed a slower B cell reconstitution potential at week 5 (p = 0.004) and a wave of increased granulocyte production at week 10 (p = 0.003). This observation is in agreement with our earlier finding that the JAM-C" population contains 32% of Lin^" /Sea- 17c-Kit⁺ cells with myeloid potential. The presence of non-self-renewing myeloid progenitors in the transferred population could lead to a transient increase in granulocytes. All of the mice receiving JAM-C¹ cells had donor-derived granulocytes at > 5 weeks and between 60 and 100% of the mice continued to have long-term granulocyte chimerism (Table 1). In contrast, only up to 20% of the mice receiving JAM-C^" cells showed long-term granulocyte chimerism at very low levels. The JAM- C¹ population is composed of HSCs that produce all blood cell lineages long-term, and non-renewing myeloid progenitors. Thus, JAM-C is a cell surface marker that characterizes progenitors within the BM with multi-lineage potential and increased self-renewal capacity. Mice deficient in JAM-C

To analyze the role of JAM-C in hematopoiesis, we obtained Jam-C^{" "} mice from Lexicon generated by deletion of exon 1 by homologous recombination (Figure 4A). Deletion of the gene was confirmed by PCR-based genotyping (Figure 4B) and the loss of JAM-C protein expression was assessed by RT-PCR. Western blot and IHC on testis tissue obtained from wild type, heterozygous and homozygous mice (Figure 4C, D, and E). Jam-C^{" "} mice were not born at a Mendelian ratio (Table 2) and mutant males were infertile, failing to produce mature spermatozoa (Figure 4E), as described previously(Gliki et al., 2004, supra). To address the question whether the low numbers of Jam-C^"'^" mice observed were caused by the postnatal death of mutants or a defect in sperm maturation in heterozygous males of spermatids carrying the null allele, we genotyped the offspring of heterozygous crosses at different developmental stages (Table 2). JAM-C homozygous mice were not found at expected frequency at 2 weeks after birth (3.8%), although they were appropriately presented before birth (El 8.5 21 .8%). Some mutant mice were cyanotic and gasping for air shortly after birth, filling the intestine with bubbles. The majority of mutant mice died within 48 hrs after birth (P2 6.7%). Examination of all major organ systems showed no obvious histologic lesions in any of the tissues examined from homozygous El 8.5 embryos, Pl pups and adult animals up to 9 months of age. Furthermore, there were no obvious histologic differences between mutant and wild type mice.

Since some mutant mice showed possible signs of anemia, we measured hematologic parameters and peripheral blood counts of E 18.5 embryos as well as mice 6 -10 weeks of age. The hemogram and red blood cell count did not show any differences between wild type and Jam-C ^" mice. Furthermore, there was no difference in the white blood cell, lymphocyte, monocyte, or platelet counts. Only adult mice showed a small but significant increase in the neutrophil count (p = 0.049) compared to wild type littermates. In summary, we did not observe any difference in blood parameters in the surviving Jam-C^" mice that could account for the phenotype observed in the neonates.

Immunophenotypic analysis of Jam-C^" mice

Since JAM-C is expressed on HSCs in the BM, we analyzed the distribution of mature blood cell lineages in the BM of adult surviving mice by flow cytometry. A mild increase in BM cellularity was detected in Jam-C ^" mice when compared to wild type littermates (p = 0.057; Figure 5A). No difference in the frequencies of mature lymphoid and myeloid cells was observed between wild type and Jam-C^" mice (Figure 5B and C). Analysis of the total number of cells in the BM showed that the number of granulocytes was increased in Jam-C mice (p = 0.009; Figure 5D), whereas the number of erythroblast and B cells was not different. Taken together with the complete blood cell count, these data show that loss of JAM-C leads to an increase of granulocytes in the BM and subsequently of neutrophils in the blood.

No abnormalities were observed in other immunologic tissues when comparing wild type to Jam-C ^ mice. Although there was a minor reduction in spleen size of homozygous animals (p = 0.064), spleen cellularity was not significantly different (Table 4). Mature T and B cells in the spleen were generated normally from HSCs (Table 4). There was no difference in thymus size and cellularity (Table 4) and all major thymus subsets were generated normally in Jam-C^" mice (Table 4). Thus, the generation of mature B and T cells is not affected in Jam-C ^" mice.

Hematopoietic progenitor population in bone marrow of Jam-C ^" mice Next we investigated the effect of JAM-C deficiency on the hematopoietic progenitors present in adult BM. We found the frequency within the BM and total numbers of the lineage negative population to be normal in surviving Jam-C ^" mice (Figure 6A, and B). Analysis of the frequency and total number of LSK cells including LT-HSC, ST-HSC, and MPP did not show any differences between Jam-C ^~'^~ and wild type animals (Figure 6C, D, and E). However, analysis of the total number of BM progenitors showed a significant increase in the number of CMPs (p = 0.032) (Figure 6E). As shown earlier the myeloid committed progenitors have the highest percentage of JAM-C expressing cells among the lineage committed progenitors (Figure I F). In contrast, the number of CLPs, which show lower percentage of JAM-C expressing cells, was not affected in Jam- C^'1' mice. Analysis of BM stem cells and progenitors showed that loss of JAM-C leads to an increase in myeloid progenitors, but is otherwise not essential for hematopoiesis.

In order to compare the colony-formation capacity of myeloid-committed precursors from wild type and Jam-C^"'^" mice we performed colony- form ing assays by plating out fresh BM cells in methylcellulose supplemented with growth factors and cytokines. Enumeration of colony growth on day 10 showed that Jam-C^"'^" derived cells had higher colony-forming ability than control cells (p = 0.034) (Figure 6F). This increase is caused by a higher number of erythroid colonies (BFU-E, p = 0.045) and possibly granulocyte-macrophage colonies (CFU-G, p = 0.078) but not by an increase of the most primitive in vitro colony-forming cell, the granulocyte-erythroid-macrophage- megacaryocyte colony (CFU-GEMM, p = 0.92). These results indicate that the loss of JAM-C leads to an increased growth advantage for more mature myeloid progenitors in vitro. Hematopoietic population in fetal liver of Jam-C^"Λ mice

During embryonic development, hematopoiesis occurs within the fetal liver (FL) and expansion of the stem cell population occurs between El 1 and El 5 (Ema, H. and H. Nakauchi, Blood, 2000. 95(7): p. 2284-8; Morrison, S.J., et al., Proc Natl Acad Sci U S A, 1995. 92(22): p. 10302-6). At El 5 HSCs begin to migrate from the liver to the BM, which then takes over as the major organ producing blood cells after birth. HSCs in the FL at El 4.5 can be identified by the surface expression of AA4.1 and Sca-1 on lineage negative cells (Jordan, C.T., et al., Exp Hematol, 1995. 23(9): p. 101 1 -5; Jordan, C.T., J.P. McKearn, and I. R. Lemischka, Cell, 1990. 61(6): p. 953- 63) and are called LSA cells (Lin^"Sca-l AA4.1⁺). The defect in the BM of Jam-C '^" mice could result from an impaired generation of fetal HSCs, defective migration to the BM or a defective bone marrow environment. We therefore analyzed the HSC compartment in fetal Jam-C ^~'^~ mice. JAM-C is highly expressed on LSA cells in the FL at E 14.5 by flow cytometry (Figure 7A). Jam-C^"'^" mice had similar numbers of total FL cells and LSA cells when compared to wild-type littermates (Figure 7B and Table 4). We found no difference in the frequency and total number of mature cells of the myeloid lineage like erythroblasts (Terl 19⁺) and granulocytes (Gr-l/CDl I b⁺) (Figure 7C, D, and Table 4). In summary, we found that Jam-C deficiency does not affect the number of HSCs and mature myeloid cells in FL, despite the high expression levels on LSA cells.

To identify functional defects in myeloid-committed progenitors in FL of Jam-C ^Λ mice, we compared their in vitro colony-formation ability. E14.5 FL derived cells were plated in methylcellulose supplemented with growth factors supporting myeloid growth. Colony counts on day 10 showed a slight increase in CFU-GM (p = 0.061) and a minor increase in total colonies (p = 0.074) that was not statistically significant (Figure 7E). Next we transplanted FL cells into lethally irradiated mice to assess whether there was an intrinsic engraftment defect. FL cells from either Jam- C^"'^" mice or wild type littermate controls gave 100% survival. There was no difference in the extent of reconstitution by FL cells between J am -C^7" mice and wild type littermate controls (Figure 7F) and all blood cell lineages were equivalent long-term (data not shown). These results show that the loss of JAM-C is not vital for myeloid progenitor development in the FL and seeding of the BM. Thus the increase in myeloid progenitors in the adult bone marrow is not likely to be the result of a developmental defect. Discussion

In the present study we show that the cell adhesion molecule JAM-C is highly expressed on HSC and plays a role in myeloid progenitor generation. Expression levels correlate with self-renewal, being highest on LT-HSCs and decreasing with differentiation. Expression is maintained longest among myeloid committed progenitors and deletion of Jam-C in mice resulted in an increase in myeloid progenitors and granulocytes in the bone marrow of adult mice. Loss of Jam-C has no effect on myeloid lineage generation in the fetal liver during development. The JAM-C⁺Lm^" population in the bone marrow is composed of HSCs that produce all blood cell lineages long-term, and non- renewing myeloid progenitors in lethally irradiated mice. These results suggest that JAM-C is a cell surface marker that characterizes early progenitors within the BM and regulates myeloid progenitor differentiation.

More recently, it has been proposed that JAM-C mediates cell polarization (Ebnet et al., 2004, supra). The defect in spermatid maturation in Jam-C^"''^" mice indicated JAM-Cs requirement for the assembly of a cell polarity complex in vivo (Gliki et al., 2004, supra). HSCs must also polarize and undergo asymmetric cell division in order to make cell fate decisions at branching points of differentiation (Ho and Wagner, 2007, supra; Wilson and Trumpp, 2006, supra). In Jam-C^"''^" mice the number of stem and progenitor cells was unaltered up to the stage of MPPs, indicating that JAM-C does not control the population size of the stem cell compartment. In contrast, the number of myeloid committed progenitors was increased, showing that JAM-C plays a role in fate decision towards the myeloid lineage. Little is known about the mechanisms by which lymphoid-myeloid fate decisions are induced. A study has shown that the gene expression program of the myeloid lineages is present in HSCs (Akashi et al, 2003. supra). It has thus been proposed that differentiation into the myeloid lineage is permissive (Laiosa, CV. , M. Stadtfeld, and T. Graf, Annu Rev Immunol, 2006. 24: p. 705- 38). In contrast, differentiation of HSCs into the lymphoid lineage is largely instructive requiring signals from the 11-7 receptor and Flt3. We postulate that loss of JAM-C mediated cell polarization may lead to stem cell differentiation. At the myeloid-lymphoid branching point this could lead to an increase in myeloid lineage commitment since HSCs already express myeloid regulators and lymphoid differentiation signals are limited. Probably additional regulatory mechanisms controlling myeloid progenitor generation and myeloid population size arc functional in the Jam-C ^" mice, preventing excessive differentiation leading to the development of myeloid proliferative disorders or a depletion of HSCs in the stem cell compartment. The exact molecular mechanisms by which JAM-C influences differentiation have not been identified. It is conceivable that JAM-C aids in the definition of cells that remain undifferentiated through adhesive mechanisms or by providing environmental polarity. During stem cell maturation JAM-C could become limiting either through decreased expression levels on the HSC itself or through loss of interaction with its ligand. JAM-C might be most critical for more mature progenitors expressing low levels of the protein and lack of JAM-C mediated interactions could at this point lead to myeloid lineage commitment. Furthermore, it is not known if the effect is mediated through JAM- C on HSCs or also through other JAM-C expressing cells. Certain aspects of stem or progenitor cell deficiencies may be concealed during steady-state hematopoiesis. Perturbation of the steady state by induction of hematological stress has the potential to uncover defects that are not normally apparent. Competitive transfers and serial transplants are experimental approaches that could help reveal further functions of JAM-C during hematopoiesis and its requirement for long-term stem cell maintenance. Due to the poor survival of Jam-C ^" mice of only 3.8%, statistically significant transfer experiments are not easily feasible.

Many studies have shown that adult HSCs in the bone marrow differ from fetal liver HSCs in their gene expression (Ivanova, N. B., et al., Science, 2002. 298(5593): p. 601 -4; Morrison, SJ. , et al., Proc Natl Acad Sci U S A, 1995. 92(22): p. 10302-6), function (Harrison and Lerner, Blood 78: 1237- 1240 ( 1991 ); Rebel, V.I., et al., Blood, 1996. 87(8): p. 3500-7), and regulation (Hock, H., et al., Genes Dev, 2004. 18( 19): p. 2336-41 ; Park, LK, et al., Nature, 2003. 423(6937): p. 302-5. In addition, the differentiation potential of fetal liver progenitors differs from those in the bone marrow (Kawamoto, H, K. Ohmura, and Y. Katsura, Int Immunol, 1997. 9(7): p. 101 1-9; Lacaud, G., L. Carlsson, and G. Keller, Immunity, 1998. 9(6): p. 827-38), implying that fetal and adult lymphoid- myeloid branching points are not equivalent (Laiosa et al., 2006, supra). In support of this idea we found myeloid progenitor generation not to be affected in the fetal liver of Jam-C^" mice, indicating different requirements for JAM-C in adult and fetal HSCs. Possibly the requirement of JAM-C in myeloid progenitor generation is restricted to HSC differentiation in the bone marrow. Furthermore, JAM-C is not required for the seeding of the BM by FL HSCs, since Jam-C^{" "} mice have normal numbers of HSCs in the marrow and transplanted Jam-C^"Λ FL cells fully reconstituted lethally irradiated mice long-term.

Since JAM-C has been implicated in leukocyte migration across endothelial boarders (Weber et al, 2007, supra; Chavakis et al, 2003, supra, Bazzoni, 2003, supra, Chavakis, T. and V. Orlova,

Methods MoI Biol, 2006. 341: p. 37-50) the increase in granulocytes could be caused by a defect in migration. For example granulocyte recruitment into peripheral tissue in response to inflammation has been shown to involve JAM-C through its interaction with Mac-1 (αMβ2; CDl l b/CD18) (Zen et al., 2004, supra, Chavakis et al., 2004, supra, Aurrand-Lions et al., 2004, supra). In our studies Jam- C^"'^" mice show a granulocyte increase in the BM. However, the number of circulating neutrophils under non-inflammatory conditions is only slightly affected, indicating that control of granulocyte egress is still functional in Jam-C ^"'^" mice. In comparison with published data (Imhof, B. A., et al., J Pathol, 2007. 212(2): p. 198-208), the neutrophilia is very moderate, possibly due to the lack of infection in our mice. This is supported by the observation of Imhof et al. that the number of circulating neutrophils is higher in moribund mice. Furthermore, mice with rescued expression of JAM-C on endothelial cells do not suffer from infections and have reduced numbers of circulating neutrophils. Interestingly, these mice still showed a trend towards increased circulating neutrophils under non-inflammatory conditions in comparison to littermate controls. Possibly, an additional defect to the one caused by loss of endothelial JAM-C, could influence the number of granulocytes in the BM and subsequently those of circulating neutrophils. It would be of interest to analyze the number of granulocytes in the BM of animals with rescued endothelial JAM-C expression or with Jam-C deficiency restricted to HCSs.

In agreement with other published data on Jam-C ^" mice, we have observed low neonatal survival and defects in sperm maturation. Some of the neonates were cyanotic and gasped for air, suggesting that the animals suffered from hypoxia. It has been published recently, that pulmonary dysfunction causes poor survival of Jam-C '^" mice and that rescue of vascular expression of JAM-C results in a better survival (Imhof et al., 2007, supra). Since the majority of mice die within 48 hours after birth and they did not show pulmonary infections or an increase in circulating neutrophils, we speculate that additional defects or differences in the mouse strains must be causing the observed neonatal death. We have ruled out the possibility of a HSC defect leading to anemia, since we did not find any differences in the hemogram of El 8.5 embryos in Jam-C deficient mice. Jam-C has been proposed as a candidate gene causing severe congenital heart defects in patients with 1 Iq terminal deletion disorder on the basis that it is located within that region and is expressed during human cardiogenesis (Phillips, H. M., et al., Genomics, 2002. 79(4): p. 475-8). Although Jam-C RNA has been detected in mouse heart, it remains to be shown whether JAM-C protein is expressed in the murine heart at different developmental stages and if loss of expression leads to a congenital heart defect in mice causing neonatal death.

Including JAM-C as a marker on lineage negative BM cells yields HSC enrichments that are comparable to previously identified markers (Okada et al., 1992) and transfer of these cells lead to long-term multilineage reconstitution in lethally irradiated mice. The identification of simple combinations of markers that allow reliable identification and purification of the most primitive

HSCs are of great interest. Using JAM-C as sole marker to purify HSCs from lineage negative BM cells yields HSC enrichments similar to the previously identified markers Sca-1 and c-Kit (Okada et al., 1992, supra). A large proportion of the sorted JAM-C' cells grow in methylcellulose and the colonies show phenotypes comparable to those grown from LSK cells. Nevertheless, there is a sub- population of JAM-C¹ cells, whose growth is not supported in the cytokines provided. JAM-C is expressed on a variety of cell types like endothelial cells (Aurrand-Lions, J Biol Chem 2001 , supra, Aurrand-Lions, Blood, 2001 , supra and fibroblasts (Morris et al., 2006, supra) which could be co- purified with lineage negative BM cells. The transfer Of JAM-C⁺ cells leads to long-term multilineage reconstitution in lethally irradiated mice. In contrast to LSK cells, repopulation leads to a temporary increase in myeloid reconstitution suggesting that in vivo expansion of the myeloid committed progenitors contained within the JAM-C⁺ population is supported. These progenitors are non-self- renewing leading to a normally sized myeloid compartment at later time points. In summary, JAM-C seems to identify HSCs in the BM having long-term repopulation potential as well as non-self- renewing myeloid potential.

In summary our studies provide the first evidence that JAM-C is expressed on HSC in the BM and plays a role in the differentiation of HSCs into myeloid progenitors. Significantly, JAM-C defines a multipotent stem cell population able to reconstitute all blood cell lineages and its loss leads to a deregulation of myeloid development. Given the wide expression of JAM-C, we expect that JAM-C may have additional functions in HSC regulation that are not revealed by the present study. Future studies will be needed to identify the specific mechanisms by which JAM-C regulates HSC differentiation.

Example 2

JAM-C expression on human hematopoietic stem cells

Materials and Methods

Purified human bone marrow derived mononuclear cells (MNC) were resuspended in HBSS containing 2% FCS. Fc receptors were blocked with anti-CD16 (3G8; BD Pharmingen, San Diego, CA) and human IgGl (Caltag Laboratories, Burlingame, CA). Surface expression of JAM-C was detected by staining with biotinylated mouse monoclonal anti-human JAM-C (MAJIRl ) followed by PE conjugated streptavidin secondary reagent (BD Pharmingen, San Diego, CA). Stem and progenitor cells were identified by staining with the APC-conjugated anti-CD34 (BD Pharmingen, San Diego, CA).

The results are shown in Figure 8. Human Bone Marrow was stained with the human stem cell marker anti-CD34 and analyzed for their JAM-C expression. Shown is a representative histogram with anti-JAM-C staining (black histogram line), isotype control (filled grey histogram).

Throughout the foregoing description the invention has been discussed with reference to certain embodiments, but it is not so limited. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An isolated bone-marrow (BM) derived hematopoietic stem cell (HSC) population enriched in progenitor cells expressing junctional adhesion molecule-C (JAM-C).

2. The HSC population of claim 1 which is lineage negative.

3. The HSC population of claim 2 comprising at least about 60% of the JAM-C expressing progenitor cells.

4. The HSC population of claim 2 comprising at least about 70% of the JAM-C expressing progenitor cells.

5. The HSC population of claim 2 comprising at least about 80% of the JAM-C expressing progenitor cells.

6. The HSC population of claim 2 comprising at least about 85% of the JAM-C expressing progenitor cells.

7. The HSC population of claim 2 comprising at least about 90% of the JAM-C expressing progenitor cells.

8. The HSC population of claim 2 comprising at least about 95% of the JAM-C expressing progenitor cells.

9. The HSC population of claim 2 comprising at least about 99% of the JAM-C expressing progenitor cells.

10. The HSC population of claim 2 comprising at least about 99.5% of the JAM-C expressing progenitor cells.

1 1. The HSC population of claim 2 comprising at least about 99.9% of the JAM-C expressing progenitor cells.

12. The HSC population of claim 1 having an increased self-renewal capacity and multi- lineage potential.

13. The HSC population of claim 1 wherein the JAM-C expressing progenitor cells comprise cells capable of reconstituting multiple blood cell lineages.

14. The HSC population of claim 1 wherein the JAM-C expressing progenitor cells comprise cells capable of reconstituting all blood cell lineages.

15. The HSC population of claim 1 wherein the JAM-C expressing progenitor cells comprise myeloid progenitor cells.

16. A composition comprising an HCS population according to any one of claims 1-15.

17. The composition of claim 16, which is a pharmaceutical composition.

18. A method of isolating a population of primitive hematopoietic stem cells (HSCs) from bone marrow (BM), comprising separating HSCs expressing junctional adhesion molecule-C (JAM-C) from the BM.

19. The method of claim 18 wherein said HSCs are isolated from a lineage negative (Lin ) fraction of the BM.

20. The method of claim 19 wherein JAM-C is used as a single marker for isolating said

HSCs.

21. The method of claim 19 wherein Sca-1 and c-Kit are used as additional markers for isolating said HSCs.

22. A method of treating a hematopoietic cancer comprising introducing into a patient in need an HSC population according to any one of claims 1 -15.

23. A method of treating cell damage caused by chemotherapy treatment comprising introducing into a patient in need an HSC population according to any one of claims 1 -15.

24. A method of treating a blood disorder comprising introducing into a patient in need an HSC population according to any one of claims 1-15.

25. The method of claim 24 wherein said blood disorder is aplastic anemia or sickle cell anemia.