WO2001087071A1 - Methods for enriching for quiescent cells in hematopoietic cell populations - Google Patents

Abstract

Described are preferred methods for obtaining a hematopoietic cell population having a large percentage of quiescent cells having a desirable engraftment phenotype. Also described are methods for inducing apoptosis in hematopoietic cells. Further described are culture mediums and cell populations useful in and resulting from methods of the invention.

Description

METHODS FOR ENRICHING FOR QUIESCENT CELLS IN HEMATOPOIETIC CELL POPULATIONS

BACKGROUND

The present invention relates generally to hematopoietic cells and in particular to methods involving the use of polypeptides to enrich hematopoietic cellular populations for quiescent cells.

As further background, the importance of the hematopoietic microenvironment (HM) in the maintenance and regulation of blood cell production is demonstrated by studies of genetic mutants which show deregulated hematopoiesis in vivo due to abnormalities in HM proteins; by the observation that bone marrow homing of hematopoietic stem cells (HSC) is required for successful blood cell production following transplantation; and by in vitro studies which have demonstrated that ex vivo maintenance of HSC is significantly improved when cells are cultured in the presence of bone marrow stroma or stromal cell lines (1) (2) (3) (4) (5) (6). The HM appears to exert its regulatory effects on HSC in multiple ways, since differential regulation of HSC function is observed following contact of these cells with both live and irradiated (7) or glutaraldehyde- fixed (8) stromal layers, and following non-contact culture of HSC with soluble stromal factors (6) (9). Analysis of these effects have demonstrated constitutive and inducible production of both membrane bound and soluble cytokines (10) (11) (12), and synthesis of adhesion molecules such as glycosaminoglycans (13), and extracellular matrix (ECM) proteins including fibronectin and the collagens (14).

ECM proteins of the HM have an important architectural role, providing the scaffolding upon which hematopoietic cells are able to interact with regulatory molecules. However, it has also been demonstrated that ligation of the receptors by which cells adhere to the various components of their microenvironment may initiate intracellular signaling (15) (16) (17) (18) (19), resulting in effects on hematopoietic cell proliferation, survival, migration and differentiation. These observations suggest that adhesive interactions may participate directly in the regulation of blood cell production, and that cell adhesion plays a more complicated role than simply locating cells in their microenvironment.

Fibronectins (FN) comprise a family of ECM proteins which are ubiquitously expressed in the HM (14) (20) (21) being found throughout bone, the central marrow and, importantly, in the endosteum, where the most primitive HSC are primarily found (22) (23). These alternatively spliced high molecular weight glycoproteins contain binding sites for heparin, collagen, fibrin and gelatin, suggesting an important architectural role for FN. In addition, FN also contains binding sites for the integrin cell adhesion molecules very late antigen-4 (VLA-4; αφi, which binds sites defined by the synthetic peptides CS1 and CS5 within the alternatively spliced IIICS region of the molecule), V A-5 (ofeβi, which recognizes the minimal binding sequence Arg-Gly-Asp (RGD) as well as two other synergistic binding sites, all of which are located in the cell-binding domain of the FN molecule), and the cell surface complex of chondroitin-sulfate proteoglycan and CD44 (which recognizes the high affinity C-terminal heparin-binding domain of FN). These FN receptors are all expressed in a functional state by HSC (24) (25) (26) (27) (28) (29) (30), suggesting that stem cell-FN interactions may also play a direct role in hematopoietic regulation at the stem cell level.

Ligation of integrin receptors by FN has been shown to evoke physiologic responses in some hematopoietic cells and cell lines, although these responses are varied. Adhesion to FN is involved in HSC migration in vitro (31), but the role of FN and its receptors in in vivo HSC homing and trafficking is not clear. β1 -integrin receptors are required for marrow localization (24) (29) (32); however, other ECM proteins in the HM may also be ligands for these receptors (33). Adhesion of cells to FN has also been shown in some cases to stimulate proliferation (16) (34) and increase cell production (35) (36) (37); however, other studies consistently demonstrate inhibition of proliferation by ligation of integrins (8) (38). In addition, while increased apoptosis in some cells has been shown following ligation of VLA-5 (39) (40), other data suggests that adhesion of HSC to FN may play a role in the survival and maintenance of human cells capable of repopulating hematopoiesis in immune deficient mice (34). The latter observation is further supported by many studies demonstrating increased gene transfer into engrafting HSC following transduction on FN (34) (41) (42) (43). Thus, published studies show differing and sometimes contradictory results. These differences may be attributable to disparity in target cell populations, culture conditions, or to crosstalk between intracellular signaling pathways elicited by dissimilar adhesive interactions and/or cytokines. Therefore, integrin- mediated hematopoietic regulatory processes appear to be complex and multifactorial.

Consequently, the exact mechanisms by which hematopoietic cell behavior is modified by integrin ligation and activation remain to be elucidated. The aim of the studies described here was to examine the biological consequences of HSC integrin-FN matrix interactions, both in terms of the early events that follow adhesion of a purified population of HSC to FN, and following culture of cells on FN over extended periods, as is required in retroviral transduction protocols. The results demonstrate that adhesion of primitive hematopoietic progenitor cells to FN matrix results in the death of a subset of these cells. However, the surviving cells, which include those with the capacity for long term hematopoietic reconstitution, are predominantly quiescent when adherent to FN, a phenotype which may be critical to retaining the in vivo homing and engraftment potential of these cells. SUMMARY OF THE INVENTION

Hematopoietic stem cells (HSC) have been cultured on a fragment of fibronectin (FN) containing a VLA-4 binding site prior to transplantation into I/1//I/IΛ mice. Transplant analysis demonstrated preferential survival of reconstituting stem cells in cultures in which adhesion to FN was present. Apoptosis and proliferation were examined in cells cultured on FN peptides. Analysis of apoptosis showed increased death of cells adherent to FN, compared to non-adherent cells. Analysis of proliferation demonstrated that while at day 1 a similar fraction of adherent and non- adherent cells was in cycle, the cells adherent to FN at day 6 were primarily quiescent. Thus, it has been discovered that adhesion of purified HSC to FN matrix via integrins results in the death of a subset of these cells. It has also been discovered that the surviving cells, which include those with the capacity for long-term hematopoietic reconstitution, are predominantly quiescent following prolonged adhesion to FN, a phenotype that is thought to be important to the retention of engraftment potential.

Accordingly, in one preferred embodiment, the invention provides a method for obtaining a population of quiescent hematopoietic cells, comprising culturing hematopoietic cells while adhering the cells to a fibronectin polypeptide so as to expand the number of hematopoietic cells, said adhering providing an increased percentage of quiescent hematopoietic cells.

Another preferred embodiment of the invention provides a method for obtaining a cell population containing quiescent hematopoietic cells comprising expanding a hematopoietic cell population while adhered to a polypeptide having a VLA-4 binding site so as to provide an increased percentage of quiescent hematopoietic cells.

In another form, the invention provides a method for inducing apoptosis of a subpopulation of hematopoietic cells comprising contacting the cells will a fibronectin polypeptide which causes apoptosis of a subpopulation of the hematopoietic cells.

In another aspect, the invention provides a medium for culturing hematopoietic cells which enriches quiescent hematopoietic cells wherein said medium comprises a fibronectin polypeptide.

The invention also provides a hematopoietic cell population enriched in quiescent hematopoietic cells, wherein the population is obtainable by a method according to the invention.

The starting hematopoietic cell population in the methods of the invention is desirably a human CD34+ hematopoietic cell population enriched in stem cells, and the fibronectin or other polypeptide is preferably a human polypeptide.

One object of the invention is to provide methods for providing hematopoietic cell populations enriched in quiescent cells.

Another object of the invention is to provide methods for treating subjects which include administering a cell population producable in accordance with the invention.

Additional objects and feature and advantages of the invention will be apparent from the disclosure herein.

DESCRIPTION OF THE FIGURES

Figure 1 : Engraftment of cells after six days culture on FN 30/35, assayed by hemoglobin electrophoresis. Lin-Sca+ cells from Hbb^s homozygous mice (Donor, D) were cultured at 5 x 10⁴ cells/35mm FN 30/35 or BSA coated well in "serum-replete" media for 6 days. The cells were then harvested, and dilutions of the wells were transplanted into recipient (R) W/W (Hbb^d/Hbb^s) mice. Results show donor cell engraftment in the recipients, assessed by analysis of hemoglobin types in the peripheral blood at 6 months. Lanes: 1 = recipient control; 2 = donor control; 3-6 = BSA, 1/8 well/mouse; 7-10 = FN 30/35, 1/8 well/mouse; Hbb-d: hemoglobin diffuse bands; Hbb-s: hemoglobin single band.

Figure 2: Effect of adhesion of Lin-Sca+Kit+ cells on survival/proliferation of HPP-CFC and LPP-CFC. Lin-Sca+Kit+ cells were cultured at 10,000 cells/35mm CH-296 or BSA coated well in "serum-free" media for 6 days. The cells were then harvested and plated in the HPP-CFC assay at 300 cells/dish. Growth factor combinations used in colony assays are shown. The results show the average cloning efficiency (± SEM) of cells from 5 experiments. No significant difference was observed between the average cloning efficiencies of either HPP-CFC (light hatching) or LPP-CFC (dark hatching) following culture on CH-296 versus BSA (P > 0.05). open boxes = cells plated in BSA cultures; solid boxes = cells plated in CH-296 cultures.

Figure 3: Reduction in cell numbers following culture of Lin-Sca+Kit+ cells on CH-296. Cell counts following culture of 10,000 Lin-Sca+Kit+ cells on CH-296 or BSA coated wells in "serum free" media. Results show the number of cells/well as percentage of input (survival), at each timepoint (average ± SEM of 6 - 11 experiments/timepoint). Open boxes = BSA culture; Solid boxes = CH-296 culture. A) 4h, 18h, 36h. B) 144h (6 days). Cell counts for CH-296 are significantly different to BSA for all timepoints measured; ^* P<0.05; ** P<0.005; *** P<0.0005. Figure 4: Death of Lin-Sca+Kit+ cells following adhesion to CH-296. Lin- Sca+Kit+ cells were cultured at 10,000 cells/35mm CH-296 or BSA coated well in "serum-free" media for periods up to 144 hours (6 days). At each timepoint, the non-adherent and adherent fractions of the wells were harvested separately, counted and stained with Annexin V-FITC and PI, prior to flow cytometric analysis. Two gatings of the files are shown: A) percentage of dying cells [Annexin V+ PI+ cells in the files (excluding small debris)], and B) percentage of early apoptotic cells [Annexin V+PI- whole cells], in the cultures. Results are the average ± SEM of 3 - 6 experiments/timepoint. Open boxes = BSA; hatched boxes = CH-296 non- adherent cells; solid boxes = CH-296 adherent cells. * P < 0.05; *^* P < 0.01.

Figure 5: Effect of adhesion to CH-296 on cell cycle status of Lin-

Sca+Kit+ cells. .Lin-Sca+Kit+ cells were cultured at 10,000 cells/35mm CH-296 or BSA coated well in "serum-free" media for A) 36 and B) 144 hours (6 days). The cells were pulsed with 10μM BrdU 1 hour prior to harvest. The non-adherent and adherent fractions of the wells were collected separately, and fixed and stained with an anti-BrdU antibody and PI, prior to flow cytometric analysis of the proportion of cycling cells in the wells. Results are the average + SEM of 3 experiments/timepoint. Open boxes = BSA; hatched boxes = CH-296 non-adherent cells; solid boxes = CH-296 adherent cells. * P < 0.05.

Figure 6: Representation of the structure of the α-chain of fibronectin and its relation to several recombinant fragments. The fibronectin type I, II and III repeats are indicated and the type III repeats numbered from 1 to 14. The three binding sites for cells are marked as CELL for cell binding domain (CBD) including the VLA-5 binding site, HEPARIN for heparin binding domain (HBD), and CS1 for the VLA-4 binding site CS1 formed by the first 25 amino acids of the alternatively spliced IIICS region. DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to certain embodiments thereof and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations, further modifications and further applications of the principles of the invention as described herein being contemplated as would normally occur to one skilled in the art to which the invention relates.

In broad aspects the present invention provides methods for obtaining hematopoietic cell populations containing in quiescent cells utilizing polypeptides containing binding sites for integrins on the cells.

The hematopoietic cell population may be obtained from any suitable source. For example, autologous or allogeneic bone marrow or autologous or allogeneic peripheral blood cells are potential sources of cell populations for human treatment or study. Bone marrow cells may be obtained for example from the iliac crest, tibia, femur, sternum, or another bone cavity. Bone marrow may be harvested by aspiration from the bone and processed as well known in the art. The marrow may be harvested from a donor, in the case of an allogeneic transplant, or from the patient in the case of an autologous transplant. Peripheral blood may also be collected from the patient or a donor in accordance with standard techniques. In this regard, known techniques for mobilizing stem cells to the peripheral blood may be used prior to collection, including the administration of cytokines to the patient or donor.

The hematopoietic cell population can be enriched in stem and progenitor cells. Known separation techniques can be used for these purposes. For example, hematopoietic progenitor cells can be separated from a general hematopoietic population on the basis of one or more cell surface antigens, illustratively CD34, CD71 or c-kit receptor, in combination with corresponding antibodies, especially monoclonal antibodies. Such separations can be conducted so as to obtain a cell population that is essentially free from mature blood cells.

A number of separation and enrichment schemes are known which utilize antibodies to such cell surface antigens. These include, for example, affinity chromatography, magnetic-based separations (e.g. using antibody-coated magnetic beads), cytotoxins, flow cytometry, and the like. The practice of such methods to obtain a cell population enriched in hematopoietic progenitor and stem cells is well within the skill of those practiced in the field.

The selected hematopoietic cell culture is contacted with a polypeptide having an integrin binding site, preferably a VLA-4 binding site and/or a VLA-5 binding site. In this regard, the VLA-4 and VLA-5 antigens are known and discussed for example in U.S. Patent Nos. U.S. Patent No. 5,583,203 issued December 10, 1996. Polypeptides having VLA-4 and VLA-5 binding sites are also known, and include for example fibronectin, which contains both a VLA-4 and a VLA-5 binding site, and vascular endothelial cell adhesion molecule (VECAM), which has a VLA-4 binding site.

Fragments of fibronectin or other polypeptides for use in the invention can be of natural or synthetic origin, and can be prepared in substantial purity from naturally-occurring materials, for example as previously described by (71)(72)(73). In this regard, reference herein to a substantially pure fibronectin or fibronectin fragments is intended to mean that they are essentially free from other proteins with which fibronectin naturally occurs. Substantially pure fibronectin or fibronectin fragments for use in the invention can also be recombinantly produced, for instance as generally described in (74)(75)(76). In particular, with reference to SEQ. I.D. NO. 3, which is an amino acid sequence for mature human fibronectin, and Figure 6, the recombinant fragments identified as H-271 (including residues Ala¹⁶⁹⁰ - _Thr ⁱ⁹⁶o _{of tne heparin} binding domain), C274 (including Pro¹²⁴³ - Asp¹⁵¹⁶ - VLA-4-site containing cell binding domain), H-296 (including Ala¹⁶⁹⁰ - Thr¹⁹⁸⁵ and having the heparin binding domain and the VLA-4 binding site), CH-271 (including Pro¹²⁴³ - Asp¹⁵¹⁶ and Ala¹⁶⁹⁰ - Thr¹⁹⁶⁰ - VLA-5-containing cell binding domain plus heparin binding site), CH-296 (including Ala¹⁶⁹⁰ - Thr¹⁹⁸⁵ and Asp¹⁹⁶¹ - Thr¹⁹⁸⁵ - VLA-5-site-containing cell binding domain plus heparin binding domain plus VLA-4 site) and C-CS1 (including Pro¹²⁴³ - Asp¹⁵¹⁶ and Asp¹⁹⁶¹ - Thr¹⁹⁸⁵ - cell binding domain plus VLA-4 binding site) may be prepared generally as described in these patents and used alone or in combination with other polypeptides in the present invention. These fragments or fragments from which they can be routinely derived are available by culturing E. coli deposited at the Fermentation Research Institute of the Agency of Industrial Science and Technology, Japan as FERM P- 10721 (H-296), FERM BP-2799 (C-277 bound to H-271 via methionine),

FERM BP-2800 (C-277 bound to H-296 via methionine), and FERM BP-2264 (H-271), as also described in (74). In addition, useful information as to fibronectin fragments utilizable herein or as to starting materials for such fragments may be found in (77), which reports further as to the above-noted recombinant fragments; in (78), which reports the structure of the human fibronectin gene; and in (79), which reports on the Heparin-ll binding domain of human fibronectin. Fibronectin fragments which contain CS-1 cell adhesion domain (VLA-4), for example as included in a 30 or 35 kd fragment (30/35 FN) and in recombinant fragments as reported in the Examples below, may be used. The skilled artisan will recognize that cell-binding activities can be provided both by the native amino acid sequences of the functional VLA- 4-binding and VLA-5-binding fibronectin domains and by amino acid sequences which differ from the native sequences yet are sufficiently similar to exhibit the cell-binding activities. These similar amino acid sequences will exhibit substantial sequence homology to their corresponding native sequences, and can include those in which amino acids have been deleted, substituted for and/or modified while nonetheless providing an amino acid sequence with the desired cell-binding characteristic. Polypeptides of the invention may be relatively short or longer, provided they contain the necessary cell-binding site(s) and activity. Typically a polypeptide having at least about 10 or 20 amino acid residues will be employed, commonly at least about 100 such residues up to about a thousand or more residues.

The pertinent biotechnological arts have advanced to a state in which the deletion, substitution, addition or other modification of amino acids in the subject functional domains can be routinely performed. The resulting amino acid sequences can then be routinely screened for the desired cell-binding activity. Given the teachings provided herein, these binding assays will represent but routine experimentation to those working in this field.

The native VLA-4 binding site of fibronectin is formed by approximately the first 25 amino acids of the of the alternatively spliced I1ICS region (Asp¹⁹⁶¹ - Thr¹⁹⁸⁵). The amino acid sequence of the native human fibronectin VLA-4 binding site is thus (SEQ. I.D. NO. 1):

Asp Glu Leu Pro Gin Leu Val Thr Leu Pro His Pro Asn Leu His Gly Pro Glu lie Leu Asp Val Pro Ser Thr

In preferred modes of carrying out the invention, a VLA-4 binding polypeptide will be used which has therein the amino acid sequence of SEQ. I.D. NO. 1 or an amino acid sequence which has at least about 70% identity to SEQ. I.D. NO. 1 , more preferably at least about 80% identity, and most preferably at least about 90% identity. In this regard, as used herein, percent is intended to mean percent identity as determined by comparing sequence information using the advanced BLAST computer program, version 2.0.8, available from the National Institutes of Health, USA. The BLAST program is based on the alignment method of (80) and as discussed in (81)(82)(83). Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the proteins being compared. Preferred default parameters for the BLAST program, blastp, include: (1) description of 500; (2) Expect value of 10; (3) Kariin-Altschul parameter λ = 0.270; (4) Karlin- Altschul parameter K = 0.0470; (5) gap penalties: Existence 11 , Extension 1 ; (6) H value = 4.94e^"324; (6) scores for matched and mismatched amino acids found in the BLOSUM62 matrix as described in (84)(85)(86). The program also uses an SEG filter to mask-off segments of the query sequence as determined by the SEG program of (87).

The amino acid sequence of the human fibronectin VLA-5 binding site is (SEQ I.D. NO. 2):

Arg Gly Asp Ser

Such sequence can be incorporated into the polypeptide, for example, by the inclusion of all or a portion of the VLA-5-containing cell binding domain of human fibronectin, or similar functional polypeptides. As with VLA-4 above, other polypeptides having amino acid sequences which similarly bind VLA-5 may also be used in the invention. In particular, while the amino acids Arg Gly Asp (RGD) have been found to be necessary for cell binding, the amino acid Ser may be substituted with other amino acids in polypeptides which nonetheless retain cell binding activity. Thus, polypeptides may be used which have the amino acid sequence Arg-Gly-Asp-X, where X is serine or another amino acid which provides cell binding activity to the sequence.

Where a polypeptide having more than one type of cell binding site is employed, adherence of the cells to cell binding sites other than that desired may be inhibited by appropriate masking or blocking of those binding sites or of the corresponding cell surface receptors. For example, the cells can be cultured in the presence of antibodies to the other receptors to inhibit or altogether eliminate the interaction of such receptors with their corresponding binding sites. Thus, as an illustration, when using a polypeptide having both a VLA-4 and VLA-5 binding site, antibodies to VLA-5 may be used to facilitate the predominance of the VLA-4 interaction and biological outcome. Such antibodies may, for instance, be monoclonal antibodies. A corresponding masking of the binding sites on the polypeptide may be achieved utilizing receptor proteins or fragments thereof.

Culture of hematopoietic cells in the presence of a polypeptide as described herein can be used to achieve a large population of adherent quiescent cells in the culture. Preferably, such culturing provide an increased percentage or number of quiescent cells as compared to that provided by a corresponding culture without the presence of the polypeptide. More preferably, the culturing will be conducted so that the percentage of adhered cells which are quiescent increases over the term of the culture, or example increasing by at least 3%, and even more desirably by at least about 5%. The resulting cell cultures can then be used for implantation in patients in need of hematopoietic reconstitution to treat an induced (e.g. by toxins or radiation in cancer therapy), congenital or genetic disorder or condition.

In addition to maintaining a large population of quiescent cells, preferred culturing conditions of the invention will also provide an expansion of the cell population over the duration of the culture period, especially in situations wherein patient engraftment with the resulting cell population is desired. For example, expansions in cell number of at least 10 fold are preferred, more preferably at least 20 fold, and most preferably at least about 30 fold.

Such cell cultures and culture methods may also be used in diagnostic assays, in the screening of pharmaceutical agents for their effect on hematopoietic cells, and in the study of proliferation and/or differentiation of hematopoietic cells. The base culture medium will be one suitable for the culture of hematopoietic cells. The culture medium can, for example, be a serum- free or serum-replete medium. Many such mediums are generally known, and their selection and use in the invention is within the purview of those working in this area. Cell culture may also be conducted in the presence of other agents known to effect cell growth or development, including for example cytokines such as stem cell factor (SCF), interleukins (e.g. IL-6, IL3), granulocyte colony stimulating factor (G-CSF), and the like.

For administration to mammals for engraftment, cells may be collected of the inventive cell cultures and loaded into a suitable device for delivery of the cells to the subject. For example, such a device may be a syringe or other container from which the cells can be transferred into the patient.

Cells cultured in accordance with the invention may also be genetically modified by known transduction techniques, including for example the use of suitable vectors such as viral vectors. In this regard, polypeptides having the Heparin II binding domain (including Ala¹⁶⁹⁰ - Thr¹⁹⁶⁰ see SEQ. I.D. NO: 3 and Fig. 6) and VLA-4 cell binding domain of fibronectin or similar sequences are known to enhance transduction by viral vectors. See, e.g. (88)(89)(90). Thus, methods of the invention may include a culture period for expansion of the cells on the fibronectin or other polypeptide containing the VLA-4 binding site, in combination with transduction with a recombinant viral vector on the same or a similar polypeptide. The genetic modification of the cells may be undertaken to cause the cells to express a protein that is missing or otherwise deficient or defective in a subject to be treated.

Illustratively, recombinant viral vectors may contain exogenous DNA and be non-pathogenic, i.e. replication-defective. These vectors efficiently transfer and precisely and stably integrate exogenous DNA into cellular DNA of host cells such as animal cells, particularly mammalian cells. For example, a nucleotide sequence including a run of bases from the coding sequence of the gene of interest can be incorporated into a recombinant retroviral vector under the control of a suitable promoter to drive the gene, typically an exogenous promoter. In this regard, the exogenous DNA can contain DNA which has either been naturally or artificially produced, and can be from parts derived from heterologous sources, which parts may be naturally occurring or chemically synthesized molecules, and wherein those parts have been joined by ligation or other means known to the art.

The exogenous DNA incorporated in the virus can be any DNA of interest for introduction into the cells. For example, the exogenous DNA can code for a protein such as adenosine deaminase (ADA) which is associated with a known disorder, or an antisense RNA, ribozyme or false primer (see e.g. (91)), for an intracellular antibody (see, e.g. (92)), for a growth factor, or the like.

As indicated, the introduced nucleotide sequence will be under control of a promoter and thus will be generally downstream from the promoter. Stated alternatively, the promoter sequence will be generally upstream (i.e., at the 5' end) of the coding sequence. In this vein, it is well known that there may or may not be other regulatory elements (e.g., enhancer sequences) which cooperate with the promoter and a transcriptional start codon to achieve transcription of the exogenous coding sequence. The phrase "under control of" contemplates the presence of such other elements as are necessary to achieve transcription of the introduced gene. Also, the recombinant DNA will preferably include a termination sequence downstream from the introduced coding sequence.

Retroviral vectors that include exogenous DNA providing a selectable marker or other selectable advantage can be used. For example, the vectors can contain one or more exogenous genes that provide resistance to various selection agents including antibiotics such as neomycin. Viral vectors used in the invention preferably exhibit the capacity to bind to an amino acid sequence of the Heparin-ll binding domain of fibronectin, including that of human fibronectin. In this regard, the capacity of a virus to bind to the amino acid sequence of the Heparin-ll binding domain and thus to serve effectively in aspects of the invention can be readily ascertained using routine procedures. Generally speaking, these assays determine the extent to which virus particles are bound to immobilized polypeptides containing the Heparin-ll binding domain, so as to resist washing from the immobilized polypeptide matrix. Briefly, for instance, a virus-containing supernatant can be incubated in a well containing immobilized polypeptide including the fibronectin Heparin-ll binding domain. The well is then extensively washed with physiologic saline buffer, after which target cells to the virus are incubated in the well to determine the level of infectious activity remaining in the well. The reduction in infectious activity, or titer, relative to the initial viral supernatant is assessed and compared to that of a similar control run (e.g. using a BSA-coated well). A significantly higher titer remaining in the Heparin-ll domain containing well as compared to the control well signifies that the subject virus is suitable for use in aspects of the invention. To facilitate this screening procedure, the viral vector may contain a selectable marker gene, as discussed above.

Using conventional procedures, cell cultures produced in accordance with the invention may be used immediately, or may be frozen, if necessary in the presence of suitable cryoprotective agents, and later thawed for use.

EXAMPLES

In order to promote a further understanding and appreciation of the present invention, the following specific Examples are provided. It will be understood that these Examples are illustrative, and not limiting, of the invention. GENERAL INFORMATION

Recombinant Fibronectin

The FN fragment FN 30/35 was prepared by chymotryptic digestion of human plasma FN and purified by gelatin-Sepharose affinity chromatography, (44) (45). FN 30/35 was used at a concentration of 75pmol/cm². Recombinant CH-296 (Takara Shuzo, Otsu, Japan) was obtained as a dry powder. It was dissolved in sterile distilled water, and further diluted in phosphate buffered saline (PBS; Gibco, Grand Island, NY). Ninety-six and 6 well non-tissue culture treated plates were coated with CH-296 at concentrations of 30 - 100nmol/cm² (29). Plates were coated by adding CH-296 or FN 30/35 to the wells, at a volume of 50μl/well of a 96 well plate or 1 ml/well of a 6 well plate, and incubating for approximately 4 hours at room temperature. The plates were then aspirated and blocked for non-specific binding with 10Oμl or 2ml

(respectively) 2% bovine serum albumin (BSA; fraction V, protease-free; Boehringer Mannheim, Indianapolis, IN) in PBS for 30 minutes. The BSA was then aspirated, and the wells washed in culture media prior to the addition of cells. Control wells were coated in tandem with 2% BSA only.

EXAMPLE 1 Isolation of Primitive Hematopoietic Progenitor Cells Female BeDa^ (C57BI/6J X DBA/2) or C57BI/6J mice were purchased from Jackson Animal Laboratories (Bar Harbor, ME), and maintained in laminar flow housing in our facility. The Laboratory Animal Resource Committee, Indiana University School of Medicine, approved all animal procedures. Bone marrow cells (BMCs) were harvested from the mice following euthanasia via CO≥ inhalation by excising the femurs, tibiae and iliac crests, and crushing them in a mortar and pestle. Crushed bones were washed repeatedly in PBS-0.1% BSA, and the resultant cell suspension was filtered through a 40μm nylon mesh cell strainer (Falcon, Becton Dickinson, Franklin Lakes, NJ). Low-density bone marrow cells (<1.083g/cm3) were isolated by discontinuous density gradient separation using Histopaque-1083 (Sigma Diagnostics Inc., St Louis, MO). Cells expressing mature cell lineage antigens were depleted by immunomagnetic selection using the MACS system (Miltenyi Biotech, Auburn, CA). The antibodies anti-B220 (CD45R; Clone RA3-6B2), anti- CD4 ( L3/T4; Clone RM4-5), anti-CD8a (Ly-2; Clone 53-6.7), anti-Gr-1 (Ly- 6G; Clone RB6-8C5) and anti-Mac-1 (CD11b; _m chain) (all from BD PharMingen, San Diego, CA) were used as a cocktail for MACS separation at optimal dilutions, pre-titered using whole bone marrow cells or spleen cells. Briefly, low density BMCs were centrifuged (1350 rpm, 5 minutes, 4°C) and the supernatant decanted. Fifty microliters of the antibody cocktail described above was added per 5x10^ cells and the antibody: cell suspension was incubated on ice for 30 minutes. The cells were then washed in PBS (no serum), and incubated with goat anti-rat IgG microbeads (Miltenyi Biotec, Auburn, CA) at 10μl beads + 90μl PBS/10⁷ cells, on ice for 15 minutes, agitating regularly. The cell-bead mixture was then washed, and resuspended in 1ml PBS-5mM EDTA-1%BSA/2x10⁸ cells. One milliliter of cell suspension was then added to the column (C column, maximum capacity 2x10⁸ cells), run into the mesh, and left to magnetise for 5 minutes. The non-magnetic fraction (mature cell lineage antigen negative cells; Lin- fraction) was collected by eluting the cells through a 22G needle with 24ml PBS-5mM EDTA-1%BSA solution. The cells were then washed in PBS-0.1% BSA.

Lin- cells were then sorted for either Sca-1+ cells (Lin-Sca+), or for Sca-1+c-kit+ (Lin-Sca+Kit+) cells. Cells were incubated with anti-Sca-1- PE (Ly6A/E; Clone E13-161.7), either alone, or with a FITC conjugated antibody directed against the c-kit receptor (CD117; Clone 2B8) (both from BD PharMingen, San Diego, CA; 10μl of each antibody diluted in 100μl PBS-0.1 %BSA/25x10⁶ cells) for 30 minutes at 4°C in the dark. They were then washed in PBS-0.1 %BSA and held on ice prior to sorting. Cells were sorted using a FACStar Plus cell sorter (Becton Dickinson, San Jose, CA), equipped with a 488nm laser (Ion Laser Technologies; Salt Lake City, UT) running at 40mW power. Unseparated bone marrow cells were used to select a blast cell light scatter region previously shown to contain the majority of hematopoietic progenitors (46). Lin- cells were then sorted within this blast cell region, to select for cells which were either positive for the Sca-1 antigen; or, using a rectilinear sort region, for cells which expressed both the Sca-1 antigen, and also the c-kit receptor (approximately 55% of the total Lin-Sca+ population; purity > 90%). Cells for sorting were kept chilled, sorted at a rate of approximately 4,000 cells/second, and either sorted directly into 96 well plates at 30 cells/well using the automatic cell deposition unit (ACDU), or collected into 8ml PBS- 0.1 %BSA. These cells were then centrifuged (1350rpm, 5 minutes, 4°C), the supernatant decanted, and the cell pellet resuspended in 300μl PBS- 0.1 %BSA prior to counting and viability determination using trypan blue (Sigma, St Louis, MO).

EXAMPLE 2 In vitro culture of sorted cells

Sorted cells were cultured in wells of 96 well (30 cells/well) or 6 well (10,000 or 5x10⁴ cells/well) plates, coated with CH-296, FN 30/35 or BSA, as described above. The cells were cultured in 200μl and 2ml

(respectively) of either "serum-free" media (X-vivo 15 Serum Free Media, BioWhittaker, Walkersville, MD), containing 1% detoxified BSA (Stem Cell Technologies, Vancouver, Canada), 2% penicillin/streptomycin (Gibco BRL, Life Technologies, Rockville, MD), 1 % glutamine (Gibco BRL, Life Technologies, Rockville, MD), and the recombinant cytokines: rat stem cell factor (rrSCF; 100ng/ml; Amgen Inc, Thousand Oaks, CA), human megakaryocyte growth and differentiation factor (rhMGDF; 50ng/ml; Amgen Inc, Thousand Oaks, CA), human interleukin 6 (rhlL-6; 50u/ml; PeproTech, Rocky Hill, NJ) and human granulocyte colony-stimulating factor (rhG-CSF; 5ng/ml; Amgen Inc, Thousand Oaks, CA); or "serum- replete" media (IMDM containing 10% bovine serum (Hyclone, Logan, UT) and the cytokines rmlL-3 (1ng/ml; PeproTech, Rocky Hill, NJ) and rrSCF (20ng/ml)). The cells were incubated at 37°C for periods of time from 4 to 144 hours (6 days). Cells in 96 well plates were counted in situ using an inverted microscope. The non-adherent and adherent fractions were harvested separately from 6 well plates. The wells were gently rinsed 3 times with the media containing the non-adherent fraction using a 2ml pipette, prior to harvesting that fraction. One ml of cell dissociation buffer (Gibco BRL, Life Technologies, Rockville, MD) was then added to the well, and the adherent cells were incubated at 37°C for 5 minutes. The cell dissociation buffer was then diluted with 1ml PBS, and the adherent cells were harvested by vigorous pipetting. The wells were rinsed with PBS, after which they were inspected visually using an inverted microscope to ensure that all of the adherent cells had been collected. The cells were then centrifuged (1350rpm, 4°C, 5 minutes), resuspended and counted.

EXAMPLE 3

Functional assays

3.1 Long term repopulatinq assay

WBB6F1/Kit Kit^w-^v

hosts) and wild type C57BI/6J mice (donors) were purchased from Jackson Animal Laboratories (Bar Harbor, ME), and maintained in laminar flow housing in our facility. Lin-Sca-1 + cells were sorted from donor C57BI/6J mice as described above, and were incubated at 5 x 10⁴ cells/well on FN 30/35 or BSA coated plates in "serum-replete" media for 6 days. At this time, the whole well was harvested, and dilutions of these cells (1/2, 1/8, 1/24 well) in 0.5ml Hanks Balanced Salt Solution containing 0.1 % (v/v) 1 M Hepes and 0.1% BSA were transplanted into recipient WA/ mice, by intravenous injection via the lateral tail vein. Animals were initially analyzed at 5 weeks pot- transplant and at monthly intervals until 7 months after transplant. At the completion of the experiment, animals were sacrificed and tissue isolated for DNA extraction. Individually marked mice were bled via the tail vein into Microtainer tubes (containing EDTA; Becton Dickinson, San Jose, CA). Analysis of donor positive peripheral blood cells was performed by cellulose acetate electrophoresis (47) to quantify increases in donor hemoglobin. The concentration of single (Hbb^s) or diffuse hemoglobin (Hbb^d) expressed as a percent of the total hemoglobin (Hbb^s plus the diffuse major and minor hemoglobin, Hbb^d) was measured with a densitometer. Southern blot analysis confirmed engraftment by donor- derived lymphoid and myeloid cells in marrow, spleen and thymus (data not shown). C57BI/6J donors are homozygous for single hemoglobin (Hbb^s/Hbb^s), and the W/W recipients are heterozygous for single/diffuse hemoglobin (50% Hbb^s/50% Hbb^d) (48).

3.2 In vitro HPP-CFC assay

Lin-Sca+Kit+ cells (purity >90%) were sorted from donor

mice and incubated at 10,000 cells/well on CH-296 coated plates in "serum- free" media for 6 days. High and low proliferative potential colony forming cell (HPP-CFC and LPP-CFC) were assayed by plating the harvested cells at low density (300 cells/dish) in a double layer nutrient agar culture system as previously described (49) (50). The recombinant cytokines murine colony-stimulating factor- 1 (rmCSF-1 ; 1600units/dish), murine interleukin 3 (rmlL-3; 200units/dish) (both Genetics Institute, Cambridge, MA) and human interleukin 1 (rhlL-1α; 1000u/dish; Genzyme, Boston, MA) were used.

3.3 Cell death analysis

Lin-Sca+Kit+ cells, sorted from B₂DeFι mice, were incubated at 10,000 cells/well on CH-296 coated plates in "serum-free" media for up to 6 days. Once harvested and counted, the cells were washed in PBS, pelleted by centrifugation (1350rpm, 4°C, 5 minutes), and resuspended in 100μl Annexin V binding buffer (10mM Hepes/NaOH, pH 7.4, 140mM NaCI, 2.5mM CaCI₂) containing 4μl Annexin V-FITC (BD Pharmingen, San Diego, CA) and 5μg propidium iodide (PI, Calbiochem, La Jolla, CA). The cells were incubated for 20 minutes at room temperature in the dark, after which an additional 200μl Annexin V binding buffer was added prior to analysis using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Cell death in the cultures was determined by measuring (i) the fraction of dying cells in the files [Annexin V+PI+ events (excluding small debris)], and (ii) the percentage of early apoptotic cells [AnnexinV+PI- whole cells] in the files.

3.4 Cell cycle analysis

B₂D₆Fι Lin-Sca+Kit+ cells were incubated at 10,000 cells/well on CH- 296 coated plates in "serum-free" media for 36 hours or 6 days. Cells were pulse-labelled with 10μM bromodeoxyuridine (BrdU) for one hour at 37°C prior to harvesting. Harvested cells were fixed in 0.5ml 0.5% paraformaldehyde (Fisher Scientific, Fair Lawn, NJ; pH 7.4, in PBS) for 5 minutes on ice. The cells were then stored at 4°C overnight prior to analysis.

Fixed cells were brought to room temperature, and then centrifuged at 1 ,350rpm for 5 minutes. The supernatant was discarded, and the cell pellet was loosened by gently vortexing. Three molar HCI containing 0.5% Tween-20 (0.5 ml, made fresh) was added, and the cells were incubated at 37°C for 30 minutes. After incubation, the cells were centrifuged, the supernatant discarded, and the cells resuspended in 0.5ml 0.1 M Na2B4.θ7, and again centrifuged. Cells were then washed in 1ml PBS, and the supernatant was decanted prior to antibody staining. The cells were incubated with 10μl FITC-conjugated anti-BrdU antibody (Boehringer Mannheim, Indianapolis, IN) diluted in 40μl PBS containing 1% Tween-20, for 30 minutes at 37°C in the dark. Following incubation with antibody, the cells were washed in PBS, and resuspended in a 5μg/ml PI solution, prior to analysis on the FACScan.

3.5 Statistical analysis

Paired students T-tests were performed using GraphPad InStat, and results were considered different if P<0.05. Results in Table 1 compared using Mann-Whitney Test. RESULTS OF EXAMPLES

Preferential survival and engraftment of transplantable stem cells after adhesion to FN 30/35

In order to investigate the effect of integrin-FN matrix interactions on the capacity of HSC to engraft and contribute to long term blood cell production following transplantation, 5 x 10⁴ C57BI/6J Hbb^s/Hbb^s Lin-Sca+ cells were cultured in "serum replete" media for 6 days on a chymotryptic fragment of FN (FN 30/35, containing the heparin binding domain and the VLA-4 integrin binding site) or BSA, prior to transplantation into

WBB6F1/Kit Kit^w-^v, Hbb^d/Hbb^s recipients. Analysis of the contribution of transplanted cells to long term blood cell production in the recipients was determined monthly by hemoglobin electrophoresis of peripheral blood. At 6 months full donor cell engraftment of 100% of mice was demonstrated following transplant of Vz well/mouse from both FN 30/35 and BSA coated plates (data not shown). No donor engraftment was seen in animals transplanted with 1/24 well/mouse. By comparison, while all mice showed either full (Figure 1 ; lanes 9,10) or partial reconstitution (Figure 1 ; lanes 7,8) following transplantation of 1/8 FN 30/35 well/mouse, no mice receiving cells from BSA-coated wells engrafted at this same dilution (Figure 1 ; lanes 3-6). When engraftment was analyzed from all animals, percent of single hemoglobin (representing donor engraftment) was not different from untransplanted controls (P>0.05, Table 1). In contrast, percent of single hemoglobin in mice transplanted with cells cultured on FN 30/35 was significantly greater than in mice transplanted with cells cultured on BSA (P<0.05). Therefore, this data demonstrates preferential survival and/or engraftment of long term reconstituting cells in cultures in which adhesion to FN is present. Culture of Lin-Sca+Kit+ cells on CH-296

Interestingly however, this data did not predict results obtained by co-culture experiments in which the HPP-CFC assay was used as functional readout of primitive hematopoietic clonogenic cells. In these studies, Lin-Sca+ cells were further purified for c-kit+ cells (Lin-Sca+Kit+), in order to increase the purity of cells reported to contain long term hematopoietic engrafting cells. In addition, the recombinant FN peptide CH-296 was used (containing, in addition to the heparin binding domain, the binding sites for both the VLA-4 and VLA-5 integrins), since multiple reports have documented that adhesion of HSC to this fragment improves gene transfer efficiency into transplantable HSC (34) (42) (43) and since we have previously demonstrated similar levels of adhesion of reconstituting cells to this fragment compared with FN 30/35 (29). Following culture of 10,000 Sca+Kit+ cells for 6 days in CH-296 or BSA- coated wells, the functional potential of the resultant cells was analyzed using the HPP-CFC assay. No significant difference was observed in either the frequency or the size (HPP-CFC vs LPP-CFC) of the colonies present in CH-296 versus BSA cultures (Figure 2), regardless of the cytokine combination used to stimulate colony growth. The discrepancy between results of this assay and those measuring transplantation potential of the cells could be due to the preferential survival of a primitive cell following culture on FN, which is capable of long term hematopoietic reconstitution, but which has poor cloning efficiency in the HPP-CFC assay; or, alternatively could be a result of FN adhesion maintaining an "engrafting phenotype" in HSC, which is lost following culture of cells in suspension (a parameter measured by in vivo, but not in vitro, assays).

In order to distinguish between these possibilities, we next investigated in more detail the events that follow adhesion of purified HSC cells to CH-296. In particular, we examined the effects of FN culture on cell death, which could lead to preferential survival of HSC on FN; and on cell cycling, which has been suggested to be associated with changes in the engrafting phenotype of primitive cells (51) (52) (53) (54). To establish the extent to which Lin-Sca+Kit+ cells adhere to FN, sorted cells were cultured in dishes coated with CH-296 or BSA for 4, 18 and 144 hours (6 days). The non-adherent and adherent fractions of the wells were then harvested separately and counted. 51.0+14.0% of these cells were adherent to CH-296 following 4 hours culture, an interaction which we have previously shown using blocking antibodies to be mediated specifically via VLA-4 and VLA-5 integrins (29). Interestingly, by 18 hours this fraction had declined to 33.8+7.0%, suggesting that extended culture on FN may reduce adhesion. However, this decline did not continue, and adhesion was maintained even following culture over 6 days (27.4+7.5% adherent cells). Non-specific adhesion to BSA in these cultures remained below 8% for each time point studied.

Cell counts obtained from these cultures demonstrated that the total number of cells in both CH-296 and BSA cultures decreased over the first 18 hours. However, a significantly greater reduction in cell numbers was observed following culture on CH-296 (Figure 3A), suggesting that adhesion of Lin-Sca+Kit+ cells to this fragment actively induces death in a subset of these cells. Reduced cell numbers were found in the CH-296 coated wells throughout the entire culture period, with significantly fewer cells in CH-296 coated wells even at 6 days (Figure 3B). This observation was not the result of losses during the harvesting process, since the reduction in cell numbers found in CH-296 versus BSA coated wells was also observed when cells were counted in situ, in 96 well flat bottomed plates using an inverted microscope. In the latter experiments, 30 Lin- Sca+Kit+ cells were deposited/well using a flow cytometer equipped with an automated cell deposition unit, cultured for 24 hours in "serum-free" media and then counted. On average, 54.5±9.8% of input cells were found in CH-296 coated wells, compared with 67.6+8.5% of input cells in BSA coated wells (n = 5, p = 0.0116).

In order to directly investigate this loss of cells cultured on CH-296, studies were done using flow cytometric analysis of Annexin V and propidium iodide (PI) staining. A higher percentage of total dying cells [Annexin V+PI+] were observed in the FN adherent versus non-adherent fraction at 18 hours (Figure 4A). In addition, at this same time point, there was an increased proportion of early apoptotic cells [Annexin V+PI- whole cells] in the FN adherent fraction (Figure 4B), although this was not statistically significant. Therefore, these results show that many cells have proceeded through the early stages of the apoptotic pathway by 18 hours of culture, and further demonstrate increased death of cells adherent to FN. A significant difference was also observed in the level of both total dying and early apoptotic cells (Figure 4A and 4B) in the FN adherent versus non-adherent fractions at 144 hours, again suggesting that adhesion to FN results in the death of a subset of these cells. Interestingly however, no significant difference was observed in the total level of cell death in the whole CH-296 versus BSA cultures (data not shown).

Cell cycle analysis of cells adherent to CH-296

It has been hypothesized that cell cycle status may affect HSC engraftment (51) (52) (53) (54). Therefore, cycle analysis of cells surviving in the CH-296 and BSA cultures was performed by measurement of BrdU incorporation after a 1 hour BrdU pulse label. Following 36 hours of culture, a similar fraction of cells in BSA cultures and FN-adherent and non-adherent cells were in cycle (Figure 5A). By comparison, analysis at day 6 demonstrated that significantly more cells in the FN-adherent fraction were quiescent compared with the non-adherent fraction

(58.3+4.1% G0/G1 phase vs. 34.0±0.5%, respectively) (Figure 5B). As seen with analysis of cell death, no significant difference was observed between the fractions of cycling cells in the total CH-296 versus BSA cultures (data not shown). Therefore, these results demonstrate that cells which survive and are adherent to CH-296 at 6 days of culture are quiescent relative to cells in the non-adherent fraction, a phenotype which correlates with enhanced HSC function in vivo (46) (55) (56). DISCUSSION OF EXAMPLES

Although recent studies have demonstrated that ligation of cell adhesion receptors by FN initiates intracellular signaling (15) (16) (17) ( 8), the varied effects of these signaling events (encompassing both stimulatory and inhibitory effects on cell proliferation, death, migration and differentiation in hematopoietic cells) suggest that adhesion-mediated regulation of hematopoietic cell function may be complex. The different responses that have been documented may be explained in part by the use of cell lines, transformed cells, lineage committed cells, or heterogeneous primary cell populations for these studies (16) (30) (40) (44) (57) (58) (59) (60). In addition, the effects elicited by adhesion to FN may vary with the developmental stage (24) (25) (28) or source of the progenitor cell population being studied (61) (62). Since cross-talk and cooperation between different adhesion and cytokine mediated signaling pathways has been documented, cellular responses are also clearly related to the specific combinations of adhesive and mitogenic interactions involved, and therefore to differences in culture conditions (11) (27) (29) (31) (36) (58) (59) (61) (63) (64) (65).

In the studies described here, we investigated the biological consequences of integrin-FN matrix interactions in hematopoietic stem and progenitor cells. Studies of functionally defined HSC investigated the survival and maintenance of engraftable cells following 6 days culture on FN 30/35. Analysis of the contribution of the cultured cells to blood cell production 6 months following transplant into W/W recipients demonstrated that, in agreement with gene transfer studies on both FN 30/35 and CH-296 (34) (41) (42), survival of reconstituting stem cells was observed in cultures where adhesion to FN was present. Interestingly however, analysis using the HPP-CFC assay, an in vitro surrogate assay for primitive hematopoietic stem and progenitor cells, demonstrated no significant difference between the frequency and proliferative capacity (as assayed by colony size) of primitive clonogenic cells following culture on CH-296 versus BSA. The discrepancy between the results obtained from these two different assay systems which has been noted in previous studies (66) (67) may be explained by (i) the preferential survival on FN of a primitive cell which is capable of long term hematopoietic reconstitution, but is not assayed in HPP-CFC cultures, or (ii) by the retention of an "engrafting phenotype" in HSC following culture on FN, a phenotype that is not measured in in vitro clonogenic assays, and which is lost to these cells following culture in suspension. In order to investigate these possibilities, we examined the early events which followed adhesion of cells to FN to determine whether adhesion perse has an effect on cell death or preferential survival of primitive reconstituting HSC or an effect on cell cycling, which may affect the engrafting phenotype of the cells. Several experimental parameters are of significance in these studies. Since it has been suggested that murine HSC divide within around 30-40 hours of in vitro culture (68), the early time points examined were prior to 40 hours. In order to examine the response of cells capable of long-term engraftment, we utilized a purified population of HSC (Lin-Sca+Kit+ cells) for these studies. In addition, we also used the recombinant FN peptide CH-296, to investigate more thoroughly its putative role in the maintenance and transduction of transplantable HSC. Although the mechanism for increased transduction of HSC on this FN fragment appears to involve co- localization of virus particles and target cells (42), the existence of a biological component to this effect has been previously hypothesized following evidence that transduction on CH-296 is associated with preferential maintenance of engraftable HSC, compared with culture of the cells in suspension (34). In the studies described here, cells were seeded at low density (~10,000/35mm well), in order to optimize study of the adhesion of progenitor cells to FN, while minimizing the effects of cell/cell interactions. The cells were cultured in serum free media (to avoid variations due to serum FN), with the cytokines SCF, MGDF, IL-6 and G- CSF at optimally defined concentrations. These cytokines, termed "early" acting cytokines (69), were chosen due to their reported ability to maintain survival of primitive HSC in vitro, and due to the ability of both SCF and MGDF to increase adhesion of hematopoietic cells to FN in culture (58) (70).

A majority of Lin-Sca+Kit-i- cells were adherent to CH-296 following a 4 hour incubation, an interaction which we have previously shown to be mediated via VLA-4 and VLA-5 (29). This adhesion appeared to result in down regulation of FN receptor function, since fewer adherent cells were observed following incubation on FN for 18 hours, compared with for 4 hours. However, the level of adhesion observed at 18 hours was maintained throughout the remainder of the culture supporting the hypothesis that integrin receptor expression/avidity is reversibly regulated (30) (31) (61) (65) (70). Cell counts from these cultures demonstrated that the total number of cells in both CH-296 and BSA cultures decreased over the first 18 hours. However, the decline in cell numbers was significantly greater in FN coated wells, and flow cytometric analysis of Annexin V and PI staining of the cells demonstrated a greater proportion of both early apoptotic and late stage dying cells in the CH-296 adherent fraction of these cultures. These results also suggest that adhesion of Lin- Sca+Kit+ cells to CH-296 may induce death in a subset of these cells. Previous studies have demonstrated cell death, particularly of mature hematopoietic subsets, following adhesion to FN (40) (39) and (Kapur and Williams, manuscript in preparation). Therefore, these results, together with the observation that engraftable HSC are maintained following culture on FN, could support the hypothesis that adhesion to FN results in selective death of relatively mature subsets in the Lin-Sca+Kit+ fraction, and in preferential survival of HSC. However, since no significant difference was observed in the frequency of LPP-CFC or HPP-CFC surviving in cultures on FN versus BSA, this interpretation appears less likely.

Alternatively, the results suggest maintenance of an "engrafting" phenotype in cells adherent to FN. Previous studies have demonstrated that quiescent cells maintain significantly higher engrafting potential than cells in active cycle (52) (53) (54). To investigate the hypothesis that maintenance of an "engraftable" HSC phenotype in cultures containing FN is related to the quiescence of these cells, the cell cycle status of FN adherent cells was compared with that of non-adherent cells. The results showed that although CH-296 adherent and non-adherent cells were cycling equally at 36 hours, by 6 days, those cells which survived and were adherent to CH-296 were quiescent relative to non-adherent cells. In the context of in vivo transplantation studies, these results provide a mechanism for the maintenance of repopulating cells reported when FN has been utilized in gene transfer protocols (34). Thus, the studies presented here, and those from gene transfer experiments (34) (41) (42) (43) suggest that engraftment of transduced cells following long term culture in the presence of FN may be related to the maintenance or re- entry of adherent cells in G0/G1. Since integration of vector sequences requires cell division, the implication of these data are that adhesion to FN maintains cells in G0/G1 after cell division in vitro.

In summary, the results described here suggest that the improved engraftment observed following 6 days of culture in the presence FN may be a consequence of the survival and quiescence of HSC, allowing the cells to retain the ability to home to the bone marrow and engraft following transplantation.

Table 1 : Densitometric analysis of different hemoglobin bands after electrophoresis

Hbb^d (%) of totaή Hbb⁵ (%) of total

C57BL6 mice 0.000 100

W/W^v mice (untransplanted) (4) 46.0±6.3 54.0±6.3^B

W/W^v transplanted with 4.97±9.9 95.0+9.9 day 0,1500 cells (4)

6 day cultured on BSA(4) 45.5+0.6 54.3+9.6

6 day cultured on 30/35(4) 13.0+15.1 86.8+15.4°

^ATotal Density = densities of single hemoglobin (Hbb^s) and diffuse hemoglobin (Hbb^d) bands. Numbers in parenthesis shows the number of animals analyzed. ^BP<0.05 vs 30/35 culture; NS versus BSA culture ^CP<0.05 vs BSA culture

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While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.

Claims

WHAT IS CLAIMED IS:

1. A method for obtaining a population of quiescent hematopoietic cells, comprising culturing hematopoietic cells while adhering the cells to a fibronectin polypeptide so as to expand the number of hematopoietic cells, said adhering providing an increased percentage of quiescent hematopoietic cells.

2. The method of claim 1 wherein said hematopoietic cells are enriched in hematopoietic stem cells.

3. The method of claim 1 or 2 wherein said hematopoietic cells are human cells.

4. The method of any of claims 1 -3 wherein said fibronectin polypeptide is a human fibronectin polypeptide.

5. The method of any of claims 1-4 wherein said culturing is effective to increase the percentage of adherent quiescent hematopoietic cells by at least about 3%.

6. The method of any of claims 1 -5 wherein said culturing is for a duration of at least about 3 days.

7. The method of any of claims 1-5 wherein said culturing is for a duration of at least about 4 days.

8. The method of any of claims 1 -7 wherein said culturing is for a duration of at least about 6 days.

9. The method of any of claims 1-8 wherein the fibronectin fragment has a VLA-4 binding site.

10. The method of any of claims 1 -8 wherein the fibronectin fragment also has a Heparin-ll binding site.

11. A method for obtaining a cell population containing quiescent hematopoietic cells comprising expanding a hematopoietic cell population while adhered to a polypeptide having a VLA-4 binding site so as to provide an increased percentage of quiescent hematopoietic cells.

12. The method of claim 11 wherein said hematopoietic cell population is enriched in hematopoietic stem cells.

13. The method of claim 11 or 12 wherein said hematopoietic cell population is a human cell population.

14. The method of any of claims 11-13 wherein said polypeptide is a fibronectin polypeptide.

15. The method of any of claims 11-14 wherein said culturing is effective to increase the percentage of adherent quiescent hematopoietic cells in the population by at least about 3%.

16. The method of any of claims 11-15 wherein said culturing is for a duration of at least about 3 days.

17. The method of any of claims 11-15 wherein said expanding comprises culturing for a duration of at least about 4 days.

18. The method of any of claims 11-17 wherein said hematopoietic cell population contains neoplastic hematopoietic cells.

19. The method of any of claims 11-18 wherein the fibronectin fragment has a VLA-4 binding site.

20. The method of any of claims 11-18 wherein the fibronectin fragment has a VLA-5 binding site.

21. A method for inducing apoptosis of a subpopulation of hematopoietic cells comprising contacting the cells will a polypeptide having a VLA-4 binding site under conditions to cause apoptosis of a subpopulation of the hematopoietic cells.

22. The method of claim 21 wherein said hematopoietic cell population is a CD34+ hematopoietic cell population.

23. The method of claim 21 or 22 wherein said hematopoietic cell population is a human cell population.

24. The method of any of claims 21 -23 wherein said polypeptide is a human fibronectin polypeptide.

25. The method of any of claims 21 -24 wherein the fibronectin fragment has a Heparin-ll binding site.

26. The method of any of claims 21 -25 wherein the fibronectin fragment has a VLA-5 binding site.

27. A method of treating a subject, comprising administering to the subject a cell population produced in accordance with any of claims 1- 20.

28. A medium for culturing hematopoietic cells which enriches quiescent hematopoietic cells wherein said medium comprises a fibronectin polypeptide.

29. A hematopoietic cell population enriched in quiescent hematopoietic cells, said cell population obtainable by a method according to any of claims 1-20.