KR20030032955A - Methods for modulating the proliferation of hematopoietic cells - Google Patents

Abstract

The present invention describes a method of proliferating erythroid progenitor cells or inducing apoptosis of erythroid progenitor cells by contacting the cells with a polypeptide having an integrin VLA-5 or VLA-4 binding site, respectively. Cell populations cultured under these conditions can be found useful in the treatment of patients with natural or induced hematopoietic deficiency in the study of red blood cell development and other fields.

Description

Methods for modulating the proliferation of hematopoietic cells

background

The present invention generally relates to methods for controlling proliferation of hematopoietic cells and, in particular, hematopoietic cells and, in particular, erythroid line specific cells, using polypeptides having VLA binding sites.

A further technical background is that the adhesion interactions between hematopoietic progenitors and hematopoietic stem cells and the hematopoietic microenvironment play an important role in maintaining the hematopoietic process (1-5). Hematopoietic growth factor is a powerful regulator of the hematopoietic process. In addition, these proteins are involved in regulating the adhesion between hematopoietic progenitor cells and extracellular matrix proteins through changes in integrin receptor activation (6-9). The role of an adhesion molecule alone or with a growth factor to maintain proliferation, differentiation and survival of hematopoietic cells is not well understood [10 and 11], but the co-operation between growth factor receptors and integrins is normal. It has been assumed to be necessary for hematopoietic development (12). In particular, integrin receptors such as very late antigen (VLA-4) and / or VLA-5 may interact with receptor tyrosine kinases in a unique manner to influence cellular responses. In this regard, various severity hematopoietic deficiencies have been demonstrated in mice deficient in receptor tyrosine kinase, c-Kit, its ligand hepatocyte factor (SCF) or β1 integrin, suggesting an important role for the normal blood development of these proteins. [References: 1, 13 and 14]. However, no studies in the past have demonstrated the nature of adhesion-inducing coordination between these β1 integrins and c-Kit with respect to proliferation and survival / apoptosis of hematopoietic cells. It is not yet known whether the integrins in hematopoietic cells that mediate these results and the various intracellular signaling cascades downstream of c-Kit are involved.

The investigation revealed that receptors of the extracellular matrix protein, fibronectin (FN), are involved in the adhesion of hematopoietic cells, including hepatocytes and progenitor cells, in the hematopoietic microenvironment (Refs. 15-20). FN is highly expressed throughout the hematopoietic microenvironment (21 and 22). FN molecules contain binding sites for heparin, collagen, fibrin and gelatin, suggesting that they play an important role in regulating the composition of the hematopoietic microenvironment. Binding of hematopoietic cells to FN is mediated by two or more integrin receptors. VLA-5 recognizes minimal binding sequence Arg-Gly-Asp (RGD) as well as two different co- synergistic binding sites, all located within the cell binding domain of the FN molecule (Refs. 23 and 24). VLA-4 binds to sequences in the IIICS region that are alternately spliced of FNs defined by synthetic peptides CS-1 and CS-5 (Refs. 25 and 26). These receptors play an important role in normal hematopoietic development (1, 27 and 28).

Mutant mice homozygous for null mutations of c-Kit or its ligand SCF die during embryonic development or shortly after birth due to severe anemia (14, 29, 30 and 31). Survival homozygous mutations of c-Kit demonstrate severe anemia and demonstrate a significant reduction in both immature erythroid progenitor cells and mature erythroid progenitor cells. Data from these mutant mice shows that c-Kit mediated signal transduction plays an important role in normal red blood cell development (29 and 30). The interaction of FN with erythrocyte cells is also believed to be essential for erythrocyte formation, in particular the final stage of erythrocyte differentiation (32, 33, 34, 35 and 36). Erythrocyte progenitors express both VLA-4 and VLA-5 integrins (37 and 38). Efficient production of mutant cells in vitro requires attachment to FN in some systems (39, 40, 41 and 42). In addition, it has been found that erythrocyte formation is completely blocked by treatment of normal mice with anti-VLA-4 antibodies (32). There is also some evidence of the synergy between c-Kit and integrin. Exposure to SCF increases adhesion of hematopoietic cells to FN by "inside-out signaling" (6, 9, 43, 44, 45, 46, and 47). Hematopoietic progenitor cells of c-Kit mutant mouse origin have been found to reduce basal adhesion to hepatocytes, and SCF induced adhesion to FN requires c-Kit receptor kinase activity. C-Kit mutant (W / W ^v ) mice injected with antibodies to VLA-4 do not migrate progenitor cells, including erythroid progenitor cells (13). Together these data suggest the role of c-Kit and integrin receptors in normal red blood cell development.

In some cellular systems, signal delivery downstream of receptor tyrosine kinases and integrins appears to simultaneously regulate cellular responses (48). In cells of non-hematopoietic origin, several models have been proposed to characterize the interaction between integrins and receptor tyrosine kinases, including regulation within receptor activation stages or downstream signal transduction cascades. In some cases, integrin mediated attachment is required for efficient delivery of signals from the cell surface to the target in the cytoplasm or nucleus. In fibroblasts, autophosphorylation of receptor tyrosine kinases can be enhanced by attachment to matrix proteins such as FN [Ref. 49 and 50]. In addition, platelet-derived growth factor receptor phosphorylation and epidermal growth factor receptor phosphorylation following growth factor treatment are greater in adherent cells compared to floating cells (51, 52 and 53). Dormant cells stimulated with growth factors or suspended cells planted on surfaces coated with matrix proteins activate the Ras / Raf / ERK and PI-3 kinase pathways (49). Co-operation between growth factors and cell adhesion was observed to activate the MAPK and PI-3 kinase cascades. Akt has been found to be co-synergistically activated by adhesion and epidermal growth factor treatment in fibroblasts. These signal transduction effects associated with increased cell survival are consistent with an important role of the PI-3 kinase / Akt pathway in cell survival (Refs. 54 and 55). Ras-Raf-ERK and PI-3 kinase sequencing appears to be the major intracellular target of c-Kit activation.

In view of the importance of c-Kit and β1 integrin signals in the development of erythrocyte cells and the known interaction between receptor tyrosine kinases and integrins in non-hematopoietic cells, we have found that simultaneous connectivity of integrins and c-Kit is intracellular. It was assumed to have a significant effect on the activation of signal transduction pathways and thus on the action of erythroid cells. Since attachment of hematopoietic cells to FN is a dynamic process involving cooperative continuous attachment and desorption (Refs. 12, 56 and 57), we specifically FN binding to VLA-4 or VLA-5 integrins. Cell cultures grown on peptides were used to investigate the effect of dynamic processes over time on EPC survival / apoptosis and proliferation.

Summary of the Invention

It has been found that recombinant peptides comprising binding sites for integrin very late antigen (VLA-4) or VLA-5 can be used to modulate the functional and biochemical performance of erythroid progenitor cells (EPCs). EPCs can be cultured in the presence of recombinant polypeptides (eg, CH271) containing only VLA-5 binding sites or c-Kit activation that significantly increases proliferation. EPCs can be cultured in the presence of recombinant polypeptides (eg, H296) comprising a VLA-4 binding site, which significantly kills cells. Activation of c-Kit increases the viability of EPCs growing on VLA-4 binding site containing polypeptides, but only some are rescued. Reduced EPC viability on H296 is associated with decreased activation of Akt, and increased proliferation of EPC at CH271 is associated with markedly increased and sustained activation of focal adhesion kinase (FAK) and extracellular regulated kinase (ERK) pathways. . Thus, adhesion induced signals derived from the linkage of VLA-4 and VLA-5 lead to prominent biological consequences in EPC, including death via VLA-4 and survival / proliferation through VLA-5.

Accordingly, one aspect of the present invention relates to a method of inducing proliferation of EPCs comprising increasing the proliferation of EPCs by culturing a cell culture comprising EPCs in the presence of a polypeptide having a VLA-5 binding site. In a preferred manner, the cultivation is carried out under conditions effective to increase the EPC by a factor of 1, more preferably about 3 or more times. The polypeptide is preferably recombinantly produced and may be, for example, a fibronectin or fibronectin fragment having a VLA-5 binding site. In addition, the polypeptide may optionally have no VLA binding site other than VLA-5, or may have another VLA binding site. In the latter case, the VLA-5 binding site preferably demonstrates an excellent effect. This may be facilitated by optionally including a culture of the antibody against the VLA determinants present on the EPC with the corresponding binding site. The cell culture advantageously consists mainly of (more than 50%) red blood cells, more preferably red blood cell progenitor cells.

Another aspect of the invention relates to a method of treating a patient, including administering to the patient (preferably a human) a cell culture grown as previously described. By way of example, the method can be used to treat patients who develop a genetic or congenital erythrocyte disorder.

In another aspect, the present invention relates to apoptosis of EPC, comprising inducing apoptosis of EPC by culturing a cell culture containing EPC in the presence of a polypeptide having a VLA-4 binding site, preferably a recombinant. Provide a way to induce. The culture is preferably performed under conditions effective to induce at least about 10% of the EPCs in the original culture, more preferably at least about 20% of the EPCs in the original culture. The polypeptide is preferably a fibronectin or fibronectin fragment having a VLA-4 binding site. In addition, the polypeptide may optionally have no VLA binding site other than VLA-4 or may have another VLA binding site. In the latter case, the VLA-4 binding site will demonstrate a dominant effect. This may be facilitated by optionally including a culture of antibodies directed against VLA determinants present on the EPC, corresponding to other VLA binding sites (non-VLA-4) present on the polypeptide. . The cell culture optionally consists mainly of erythroid cells, eg erythroid progenitor cells.

The invention also provides a cell composition obtainable by the method of the invention.

In a further aspect, the present invention provides a hematopoietic cell culture medium for increasing the proliferation of erythrocyte progenitor cells comprising a polypeptide having a VLA-5 binding site and an erythrocyte progenitor cell comprising a polypeptide having a VLA-4 binding site. Provided is a medium for culturing hematopoietic cells that induces apoptosis.

One object of the present invention is to provide a method for effectively propagating EPCs and thus cultured EPC containing cultures.

It is another object of the present invention to provide a method for effectively inducing apoptosis of EPC and thus cell culture.

Another object of the present invention is to provide a method of treating a patient, comprising administering an EPC containing cell culture prepared according to the present invention.

Further objects as well as features and advantages of the present invention will become apparent from the following description.

Brief description of the drawings

Figure 1 (A). Schematic description of the fibronectin peptide used in the Examples.

FN consists of a series of I, II and III repeats. Regions of FN with cell binding activity alternating RGS containing cell binding domains recognized by integrin VLA-5, non-integrin dependent high affinity heparin binding sites (HEP) and integrin VLA-4 (CS-1) It is shown as a non-III connecting segment (III CS) splice. Recognition sites for integrins VLA-4 and VLA-5 as well as heparin binding sites on the FN are indicated by arrows. Expression of integrin and c-Kit on G1E-ER2 cells. (B) G1E-ER2 cells are stained with anti-Kit-PE and analyzed by flow cytometry. Thin lines indicate background staining levels observed with a suitable isotype control antibody. Thick lines indicate c-Kit expression levels. (C) G1E-ER2 cells are stained with anti-VLA-4-FITC and analyzed by flow cytometry. Thick lines indicate VLA-4 expression levels. (D) G1E-ER2 cells are stained with anti-VLA-5-PE and analyzed by flow cytometry. Thick lines indicate VLA-5 expression levels.

Figure 2. Comparison of Adhesion Effects to FN Peptides on Proliferation of G1E-ER2 Erythrocyte Progenitor Cells with or Without SCF

Cells are incubated for 48 hours on BSA- or FN-peptide-coated dishes mediating adhesion via VLA-4 (H296) or VLA-5 (CH271) in the absence (A) or presence (B) of SCF. Proliferation is measured by thymidine incorporation assay. Bars represent mean thymidine incorporation rate (counts per minute [cpm]) (± SEM) for 6 different experiments performed in 6 replicates. Compare the scale difference between panels A and B. Panel A ( ^* ) p <0.05 VLA-4 (H296) vs VLA-5 (CH271) and BSA. Panels B ( ^* ) p <0.05 VLA-4 (H296) vs VLA-5 (CH271) and BSA; ( ^** ) p <0.05 VLA-5 (CH271) vs BSA.

Figure 3. FAK and MAP-kinases (ERKs) after integrin-mediated attachment.

Factor-deficient G1E-ER2 cells are cultured on FN peptides in the presence of SCF and analyzed at defined times. (A) Cell lysates are harvested and analyzed by Western blot with anti-FAK antibody. The lower panel shows the location of phosphorylated (pFAK) FAK and non-phosphorylated (FAK) FAK. The top panel (bar) demonstrates the relative phosphorylation of the FAK (takes 48 hour time point as 100). Bars represent mean relative phosphorylation (± SEM) for three different experiments. ( ^* ) p <0.05 VLA-5 (CH271) vs VLA-4 (H296). G1E-ER2 cells deficient in (B and C) factors are left unstimulated or mediate adhesion via BSA (B) or VLA-4 (H296) and VLA-5 (CH271) in the presence of SCF. Culture on a given FN peptide (C). Cell lysates are harvested and analyzed by Western blot with rabbit anti-phospho-ERK antibodies that specifically detect phosphorylated Thr202 and Tyr204. The lower panel shows the total Erk in each lane. The positions of phosphorylated ERK-1 (pErk-1) and ERK-2 (pErk2) are shown. Top panel (bar) shows relative phosphorylation of ERK-1 and ERK-2 at residues Thr202 and Tyr204. For phosphorylation of ERK-1 and ERK-2 after 10 minutes (panel B) (take level 100 at 10 minute time point) and 120 minutes (panel C) (take level 100 at 120 minute time point) Provide relative data. Bars represent mean relative phosphorylation (± SEM) for three different experiments. ( ^* ) p <0.05 VLA-5 (CH271) vs VLA-4 (H296). (D) G1E-ER2 erythrocyte progenitor cells are cultured on BSA or FN peptide H296 or CH271 in the presence of SCF and MEK inhibitors (PD98059). Proliferation is measured using the thymidine incorporation assay. Bars represent inhibition in three independent propagation (± SEM) experiments performed in 6 replicates. ( ^* ) p <0.05 VLA-4 (H296) to VLA-5 (CH271) and BSA; And ( ^** ) p <0.05 VLA-4 (H296) and VLA-5 (CH271) vs. BSA.

4. Linkage via VLA-4 induces cell death in G1E-ER2 cells.

G1E-ER2 erythrocyte progenitor cells were cultured on FN peptides that mediate adhesion to BSA, or VLA-4 (H296) or VLA-5 (CH-271) for 48 hours in the absence of SCF (A) or in the presence of SCF (B) . do. Cell death is quantified using staining with Annexin V and propidium iodide (PI) as described in Materials and Methods. Bars represent the percentage of total cell death (± SD) in two independent experiments performed in three replicates. ( ^* ) p <0.05 VLA-4 (H296) vs VLA-5 (CH271) and BSA.

Inhibition of Akt activation via VLA-4 enhances cell death in G1E-ER2. (A) Reduction of Akt activation in Ser473 via VLA-4 (H296) attachment. Factor deficient G1E-ER2 cells are left unstimulated or cultured on FN peptides that mediate adhesion via VLA-4 (H296) or VLA-5 (CH271) at various times in the presence of SCF. At various time points, cell lysates are then harvested and analyzed by Western blot with a rabbit anti-phospho-Akt antibody that specifically detects phosphorylated Ser473. Bottom panel shows total Akt in each lane. The position of activated Akt (pAkt) is shown. The top panel (rod) quantitatively shows the relative phosphorylation of Akt. After 120 minutes (taken as 100) data are provided relative to phosphorylation of Akt. Bars represent mean relative phosphorylation (± SEM) in three or more independent experiments. ( ^* ) p <0.05 VLA-4 (H296) vs VLA-5 (CH271). (B) G1E-ER2 cells are incubated for 48 hours on BSA or FN peptide CH271 in the presence of SCF and PI-3 kinase / Akt inhibitor (Wartmannin). Cell death is quantified by staining with Annexin and propidium iodide (PI) as described in Materials and Methods. Bars represent the percentage of total cell death (± SD) in two independent experiments performed three times. ( ^* ) p <0.05 BSA, VLA-5 (CH271) (Wartmannin) vs. BSA, VLA-5 (CH271) (without inhibitor).

Figure 6: A description of the structure of the α chain of fibronectin and its relationship with various recombinant fragments

Type I, II and III repeats of fibronectin are shown and the Type III repeats are numbered 1-14. The first of the IIICS regions that are alternately spliced and represented as CELL for the cell binding domain (CBD) comprising three binding sites for the cells, ie VLA-5 binding site, HEPARIN for the heparin binding domain (HBD) The VLA-4 binding site CS1 formed by 25 amino acids is referred to as CS1 .

Description of the preferred embodiment

To help understand the principles of the present invention, certain aspects are now cited by reference and special language will be used to describe them. Nevertheless, this is not intended to limit the scope of the present invention, and modifications, further modifications, and further applications of the principles of the present invention described herein are contemplated as would normally occur to those skilled in the art to which the present invention relates. Will be understood.

In a broad aspect, the present invention provides a method of inducing death or proliferation of erythroid lineage hematopoietic cells using a polypeptide containing a binding site for each of VLA (very late antigen) -4 or VLA-5.

Hematopoietic cell populations containing red blood cell progenitor cells (EPCs) can be obtained from any suitable source. For example, autologous or allogeneic bone marrow or autologous or allogeneic peripheral blood cells are a potential source of cell populations containing human erythrocyte progenitor cells for human treatment. Bone marrow cells can be obtained, for example, from iliac tombs, tibia, femurs, sternum or other bone cavities. Bone marrow can be harvested by inhalation from bone and can be treated as is well known in the art. Bone marrow can be harvested from a donor in case of allogeneic transplant or from that patient in case of allograft. Peripheral blood can also be collected from a patient or donor according to standard techniques. In this regard, known techniques for transferring hepatocytes to peripheral blood, including administering cytokines to the patient or donor, can be used prior to collection. have.

The hematopoietic cell population may optionally be enriched in erythroid cells and in particular erythrocyte progenitor cells. Known separation techniques such as centrifugation and / or sorting procedures can be used for this purpose. For example, white blood cells can be separated by fractional centrifugation. Hematopoietic progenitor cells can be isolated from the general hematopoietic population based on one or more cell surface antigens, such as CD34, CD71 or c-Kit receptors, in combination with corresponding antibodies, in particular monoclonal antibodies. This separation can be performed to obtain cell populations that are essentially free of mature blood cells.

Numerous separation and concentration schemes using antibodies to such cell surface antigens are known. This includes, for example, affinity chromatography, magnetic-based separation (eg, using antibody-coated magnetic beads), cytotoxicity and flow cytometry, and the like. The implementation of this method for obtaining a population of cells enriched in hematopoietic progenitor and hepatocytes is within the skill of one of ordinary skill in the art.

After enriching the hematopoietic stem cells and progenitor cells, the cell population may be further processed to enrich the erythroid cells and in particular the erythrocyte progenitor cells (BFU-E) in the cell population. For example, the cell population can be contacted with lectins, such as the Ulex europaeus flocculator described in Document 70. Such techniques can be used to result in populations containing at least about 60% and even about 80% to about 100% erythroid progenitor cells.

Alternatively, established cell lines of erythroid progenitors can be used. For example, one known cell line, designated as (G1E-ER2), is similar to primary cells in the progenitor developmental stage. These cells are similar to primary erythroid progenitor cells in terms of globin and erythrocyte specific transcription factor expression and differentiation (62 and 63). It has also been found that G1E-ER2 cells express c-Kit, VLA-4 and VLA-5 and attach to FN peptides at levels similar to primary erythroid progenitor cells.

The selected hematopoietic cell culture is contacted with a polypeptide having a VLA-4 binding site and / or a VLA-5 binding site. VLA-4 and VLA-5 antigens are known and are discussed, for example, in 71. Polypeptides having both VLA-4 and VLA-5 binding sites are also known, for example fibronectin containing both VLA-4 and VLA-5 binding sites, and vascular endothelial cell attachment with VLA-4 binding sites. Molecule (VECAM).

Fragments of fibronectin and other polypeptides for use in the present invention may be of natural or synthetic origin, for example from naturally occurring materials as described previously in references 72, 73 and 74. It can be prepared in substantial purity. In this regard, reference to virtually pure fibronectin or fibronectin fragments herein means that they are essentially free of other proteins that occur naturally with fibronectin. In addition, substantially pure fibronectin or fibronectin fragments for use in the present invention can be prepared recombinantly, for example, as generally described in references 75, 76 and 77. In particular, with respect to SEQ ID NO: 3 and Figure 6 which is the amino acid sequence for mature human fibronectin, H-271 (including residues Ala ¹⁶⁹⁰ -Thr ¹⁹⁶⁰ of the heparin binding domain), C274 (Pro ¹²⁴³ -ASP ¹⁵¹⁶ -VLA-4-site) Containing cell binding domains), H296 (including Ala ¹⁶⁹⁰ -Thr ^{1985 and} having a heparin binding domain and VLA-4 binding site), CH271 (Pro ¹²⁴³ -Asp ¹⁵¹⁶ and Ala ¹⁶⁹⁰ -Thr ¹⁹⁶⁰ -VLA-5 Cell binding domain and heparin binding site), CH296 (including Ala ¹⁶⁹⁰ -Thr ¹⁹⁸⁵ and Asp ¹⁹⁶¹ -Thr ¹⁹⁸⁵ -VLA-5-site containing cell binding domain and heparin binding domain and VLA-4 site) and CCS1 Recombinant fragments identified as (including Pro ¹²⁴³ -ASP ¹⁵¹⁶ and Asp ¹⁹⁶¹ -Thr ¹⁹⁸⁵ -cell binding domains and VLA-4 binding sites) can generally be prepared as described in the above patents and alone or It can be used in combination with other polypeptides of the present invention. The fragments or fragments from which the fragments can typically be derived are described in FERM P-10721 (H296), FERM BP-2799 (methionine) in H271 at the Institute of Fermentation Research Institute of the Agency of Industrial Science and Technology, Japan. Bound C277), FERM BP-2800 (C277 bound to H296 via methionine) and FERM-2264 (H271) and also described in E. E. coli can be obtained by culturing. In addition, useful information regarding fibronectin fragments or starting materials for such fragments that can be used herein are further reported on the above-mentioned recombinant fragments [78] and which report the structure of the human fibronectin gene. References 79 and literature 80 which reports on the heparin-II binding domain of human fibronectin can be found. For example, fibronectin fragments containing 30 or 35 kd fragments (30/35 FN) and CS-1 cell binding domains contained within the various recombinant fragments described in the Examples below can be used. Those skilled in the art will appreciate that the essential cell-binding activity is both unique to the functional VLA-4-binding and VLA-5-binding fibronectin domains and to amino acid sequences that are sufficiently similar to, but sufficiently similar to, the cell binding activity. It will be appreciated that it may be provided by. Such analogous amino acid sequences may include sequences that exhibit substantial sequence homology with the corresponding unique sequence and that provide amino acid sequences with the desired cell binding properties despite the deletion, substitution, and / or modification of amino acids.

In this regard, appropriate biotechnology techniques have advanced to the state where deletions, substitutions, additions or other modifications of amino acids in the functional domain can be routinely performed. The amino acid sequence obtained can then be routinely screened for the desired cell-binding activity. Given the teachings provided herein, such binding assays will be routine experimentation for researchers in the art.

The unique VLA-4 binding site of fibronectin is formed from approximately the first 25 amino acids of the alternately spliced IIICS region. Thus, the amino acid sequence of the unique human fibronectin VLA-4 binding site is SEQ ID NO: 1

Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu His Gly Pro GluIle Leu Asp Val Pro Ser Thr

In a preferred manner of carrying out the invention, the VLA-4 binding polypeptide having an amino acid sequence of at least about 70%, more preferably at least about 80% and most preferably at least about 90% identical to the amino acid sequence of SEQ ID NO: 1 or to SEQ ID NO: 1 Can be used. In this regard, as used herein,% means% identity determined by comparing sequence information using an improved BLAST computer program version 2.0.8, available from the National Institutes of Health. The BLAST program is based on the alignment method according to Literature 81 and discussed in Literature 82, 83 and 84. Briefly, the BLAST program defines identity as the number of identical aligned symbols (ie, nucleotides or amino acids) divided by the total number of symbols of the shorter of the two sequences. The program can be used to determine percent identity over the entire length of the protein to be compared. The preferred default parameter blastp for the BLAST program is (1) 500 descriptions; (2) an estimate of 10; (3) Karlin-Altschul parameter λ = 0.270; (4) Karlin Altschule parameter K = 0.0470; (5) gap penalty: presence 11, extension 1; (H) value = 4.94e ^-324; (6) scores for matched and mismatched amino acids found in the BLOSUM62 matrix as described in references 85, 86 and 87. The program also uses an SEG filter that masks off the segment of the query sequence as measured by the SEG program of reference (88).

The amino acid sequence of the human fibronectin VLA-5 binding site is as follows:

Arg Gly Asp Ser

Such sequences can be integrated into a polypeptide, for example, by including some or all of the VLA-5-containing cell binding domains of human fibronectin or similar functional polypeptides.

As in the case of VLA-4, another polypeptide having an amino acid sequence that binds similarly to VLA-5 can also be used in the present invention. In particular, while the amino acid Arg Gly Asp is found to be essential for cell binding, the polypeptide may retain cell binding activity even though amino acid Ser is substituted for another amino acid in the polypeptide. Thus, polypeptides having the amino acid sequence Arg-Gly-Asp-X (where X is serine or other amino acid that provides cell binding activity to the sequence) can be used.

The polypeptides used in the present invention may contain no VLA binding sites and / or other cell binding sites other than the VLA-4 or VLA-5 binding sites (ie, VLA-4 or VLA-5 is the only one on the polypeptide. Cell binding site). Alternatively, the polypeptides used may have other cell binding sites, but VLA-4 or VLA-5 binding sites may have a predominant effect on hematopoietic cells. Having a number of VLA-4 or VLA-5 binding sites, eg, from 1 to about 10 VLA-4 or VLA-5 binding sites, providing an increased number of ligands for interaction with the corresponding receptors Polypeptides may also be used.

When polypeptides having more than one kind of cell binding site are used, the attachment of unwanted cells to the cell binding site can be inhibited by appropriately masking or blocking the binding site or the corresponding cell surface receptor. For example, cells can be cultured in the presence of antibodies to the remaining receptors to inhibit or completely eliminate the interaction of these receptors with their corresponding binding sites. Thus, for example, when using polypeptides having both VLA-4 and VLA-5 binding sites, antibodies to VLA-4 can be used to promote predominant VLA-5 interactions and biological outcomes. On the other hand, predominant VLA-4 interactions and biological results can be facilitated by incorporating antibodies into VLA-5 in the culture medium. Such antibodies can be, for example, monoclonal antibodies. The binding site on the polypeptide can be correspondingly masked using a receptor protein or fragment thereof.

Culture of hematopoietic cells in the presence of a polypeptide containing a VLA-4 binding site can be used to achieve apoptosis of at least about 10% EPC in the original culture. More preferably, such culture may cause apoptosis of at least about 20% EPC and most preferably at least about 30% EPC. This culturing process may be useful for treating mixed hematopoietic cell populations to reduce the number of red blood cell lines and to correspondingly enrich other hematopoietic cell lines. The resulting cell culture can then be used for transplantation in patients in need of hematopoietic reconstitution to treat induced congenital or genetic disorders. Such cell cultures and culture methods can also be used in diagnostic assays and screening of pharmaceutical preparations for their effects on hematopoietic cells, eg, screening of agents that inhibit or prevent apoptosis of red blood cells.

Incubation of hematopoietic cells in the presence of a polypeptide having a VLA-5 binding site can be used to propagate EPC in culture to increase the original number of EPCs by one or more times. More preferably, this culturing can be carried out under conditions and for periods sufficient to increase EPC by at least two times and more preferably at least about three times. As shown in the Examples below, in the studies so far, EPCs have increased by about 5 or 6 times in culture.

The substrate culture medium may be a medium suitable for culturing hematopoietic cells. The culture medium may be, for example, a serum free or serum rich medium. Many such media are generally known and their selection and use in the present invention is within the authority of researchers in the art. Cell culture also affects cell proliferation or development, including, for example, hepatocyte factor (SCF), interleukin (eg IL-6, IL-3), granulocyte colony stimulating factor (G-CSF), and the like. It can be carried out in the presence of other factors known to affect.

When administered to a mammal for engraftment, the cells of the cell culture of the invention are collected and packed into a device suitable for cutting the cells into the subject. For example, such a device may be a syringe or other container into which cells may be delivered to a patient.

The cell populations produced according to the invention, preferably containing erythroid progenitor cells, can be implanted into a patient, alone or in combination with other cell populations. For example, a cell population highly enriched with hepatocytes or other line-unrelated cells (eg, cells of the leukocyte lineage) can be co-administered with a red blood cell-enriched population that has been propagated in accordance with the present invention.

Increased cell cultures of the invention containing EPCs can be administered to mammals (including humans) with reduced levels of erythrocyte progenitors and / or mature erythrocytes. Increased cell populations of the present invention containing erythrocyte progenitor cells can be used in the treatment of erythrocyte aplasia or anemia. This condition can be caused by disease, toxins or radiation as well as genetic or birth defects.

Cell populations of the invention containing EPCs can also be used in diagnostic assays of erythrocyte progenitor cells, for example, cells that develop in a pathological condition such as anemia or polycythemia. In addition, the cultures and culture methods of the present invention can be used in the study of the development of red blood cells, including aspects such as differentiation and proliferation. For example, cell cultures of the invention can be measured in contact with biological factors or pharmaceutical agents associated with proliferation, differentiation, cell death (including apoptosis).

By using conventional methods, the cell cultures produced according to the present invention can be used immediately or later thawed if necessary for freezing and use in the presence of a suitable cryoprotectant.

Example

To aid further understanding and evaluation of the present invention, the following specific examples are provided. These examples are illustrative but will be understood not to limit the invention.

General information

Cell line

G1E-ER2 cells have been described above and are obtained from Dr. Mitch Weiss (People Developmental Studies, Boston, Miami). Unless otherwise specified, G1E-ER2 cells contain 15% heart-inactivated embryonic hepatocyte (ES) serum from Hyclone, Logan, UT, recombinant Epo (2U / mL) from Amgen, Thousand Oak, CA Iscove's Modified Dulbecco's Medium (IMDM) containing recombinant rat (rr) SCF (50 ng / ml) from Amgen, Thousand Oak, CA (manufactured by GIBCO / BRL) , Gettysburg, Maryland). For deficiency experiments, cells are washed three times in IMDM and then resuspended in the same medium containing no serum and growth factors for 6-8 hours.

Antibody and Flow Cytometry Analysis

Red blood cells (PE) -conjugated monoclonal antibodies (MoAbs) are directed against c-Kit and VLA-5. Fluorescent isothiocyanate (FITC) -conjugated antibodies are directed against VLA-4. All PE and FITC-conjugated MoAbs, including isotope regulatory antibodies, are purchased from Pharmingen (San Diego, CA). G1E-ER2 cells (1 × 10 ⁶ ) are incubated with 1 μg primary MoAb at 4 ° C. for 30 minutes. Cells were washed three times with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) (Sigma, St. Louis, MO), and a fluorescently-activated cell sorter (FACS, manufactured by Becton Dickinson) , San Jose, CA).

Example 1

Cell adhesion assay demonstrating expression of c-Kit, VLA-4 and VLA-5 in erythroid progenitor cells

1.1 method

Recombinant human FN peptides H296 and CH271 (FIG. 1A) are obtained from Takara Shuza CO., Ltd., Japan. Non-tissue culture 6-well plates are coated overnight at 100 nmol / cm ² with FN fragments diluted in phosphate buffered saline (PBS) as described previously (18). In addition, the inventors have already demonstrated that attachment to these various recombinant FNs is specifically mediated through their integrin receptors VLA-4 and VLA-5 in hematopoietic cells (Ref. 18). To mask nonspecific binding sites, plates are incubated with 2% bovine serum albumin (PBS) in PBS for 30 minutes. The wells are then washed three times with PBS. 2 × 10 ⁶ factor-deficient G1E-ER2 cells are attached to the FN peptides over various time at 37 ° C. in the presence of 10 ng / ml of rrSCF. After incubation, the plates are carefully washed with medium to collect non-adherent cells and the cells are counted.

1.2 Results

To study the potential interactions between c-Kit and integrin VLA-4 and VLA-5, we used anti-c-Kit and anti-VLA-4 and anti-VLA-5 MoAbs coupled to FITC or PE. The flow cytometry analysis used confirmed c-Kit, VLA-4 and VLA-5 expression at the erythrocyte cell surface. G1E-ER2 cells uniformly express c-Kit (FIG. 1, Panel B), VLA-4 (Panel C) and VLA-5 (Panel D). 100% of G1E-ER2 cells express integrin VLA-4 and VLA-5 (FIG. 1; Panel C and Panel D). In order to confirm that VLA-4 and / or VLA-5 mediate adhesion of G1E-ER2 cells to FN, we applied VLA-4 (H296) or VLA-5 (CH271) (FIG. 1A) [Ref. 11 and 18, two recombinant peptides containing a single binding domain are used. G1E-ER2 cells are plated on FN-H296 or CH271 and adhesion is measured over 2 hours. Significant attachment to the FN fragment is observed by VLA-4 or VLA-5. Up to 68% of GIE-ER2 cells attached to CH271 via VLA-5 and 80% of GIE-ER2 cells attached to FN-H296 via VLA-4 (data not shown). Previous studies using primary erythroid progenitors have shown similar levels of FN as well as attachment to FN peptides (58). In contrast, the majority of cells (85%) were in suspension in BSA-coated dishes. In previous studies using these same fragments and other hematopoietic cell lines or primary cells, we presented the specificity of the attachment in these fragments in which the antibody was blocked (Ref. 18). Since the majority of EPCs are attached to FN peptides and previous studies have shown that the attachment of hematopoietic cells to FN is a dynamic process, in subsequent studies we have applied cell cultures cultured in H296, CH271 or BSA coated dishes. The effect of this method on the test was examined.

Example 2

Effect of FN on Proliferation and Survival of G1E-ER2 Cells

2.1 method

The effect of FN peptides H296 and CH271 and SCF on proliferation of G1E-ER2 cells is assayed using thymidine incorporation. 96-well non-tissue culture plates are coated with FN peptide as described above. Growth factor deficient G1E-ER2 cells are plated for 48 hours at ⁵ × 10 ⁴ cells per well with or without 100 ng / ml rrSCF. Subsequently, 1.0 uCi of [ ³ H] = thymidine (Amersham) is added to each well at 37 ° C. for 6-8 hours. Cells are then harvested using an automatic cell harvester (96 well harvester, Brandel, Gettysburg, MD) and the thymidine incorporation rate is measured with a scintillation counter. The effect of FN peptides and SCF on cell death (apoptosis and necrosis) of G1E-ER2 cells was determined by annexin-FITC and propidium-iodide according to the manufacturer's instructions (Pharmingen, San Diego, CA). Assay by staining with (PI). 24-well non-tissue culture plates are coated with FN peptides CH271 and H296 as described above. Growth factor deficient G1E-ER2 cells are plated for 48 hours at ⁵ × 10 ⁵ cells per well with or without 100 ng / ml rrSCF. Subsequently, cells are harvested, stained with Annexin-FITC and PI and analyzed by flow cytometry.

2.2 Results

2.2.1 Cooperation between c-Kit and VLA-5 Enhances Proliferation of G1E-ER2 Cells

Although the role of integrins in erythrocyte proliferation is not defined, studies have suggested that FN is required both in vitro and in vivo to provide suitable infusions for erythrocyte development [34, 35, 36, 37, 38, 39 and 40]. To determine if integrin is an important mediator of mitosis in erythrocytes, we measure DNA synthesis in G1E-ER2 cells cultured on FN peptides in the presence or absence of SCF. 48 hours after incubation on FN peptide, [ ³ H] thymidine is added for 6 hours and [ ³ H] thymidine incorporation is measured. In experiments where no SCF was added to the culture, factor-deficient G1E-ER2 cells cultured on BSA or CH271 showed similar proliferative responses (11701 ± 1257 vs. 10085 ± 1108 counts [cpm] per minute, average ± SEM, p> 0.05) (FIG. 2; Panel A). In contrast, cells cultured on H296 result in less than 50% DNA synthesis (5689 ± 614 cpm, p <0.05 vs CH271) (FIG. 2; Panel A).

In experiments in which SCF was added to the culture, G1E-ER2 cells cultured on FN-CH271 (mediated attachment via VLA-5) showed significantly higher proliferation compared to SCF stimulated cells in suspension (BSA). (231,301 ± 12114 vs. 145,173 ± 10832 cpm, mean ± SEM, p <0.05, respectively) (FIG. 2; Panel B). In contrast, even in the presence of SCF, the culture of these cells on H296 is associated with reduced proliferation compared to cells cultured in suspension (98,070 ± 5320 vs 145,173 ± 10832cpm, mean ± SEM, p <0.05, respectively). Cell cycle analysis performed by flow cytometry analysis after staining with PI demonstrated increased influx of cells to S + G2 / M in culture in which G1E-ER2 cells were grown on CH271 (data not shown) ). As such, these studies demonstrate that different biological results are stimulated by adhesion of red blood cells to FN through VLA-4 as compared to VLA-5.

2.2.2 Effect of FN on ERK, Akt and FAK Signal Delivery Pathways in G1E-ER2 Cells

Activation of MAP-kinases (ERK-1 and ERK-2) is measured using phospho-specific ERK antibodies (New England Biolabs, Beverly, Mass.). Such antibodies detect ERK only when they are catalytically activated by phosphorylation. Activation of Akt is measured using phospho-specific Akt (Ser473) antibody (New England Biolabs). Activation and expression of FAK is measured using anti-FAK antibodies (Upstate Biotechnology). All antibodies are used at 1: 2000 dilution. Briefly, non-tissue culture 6 well plates are coated with FN fragments as described above. Factor-deficient 5-8 × 10 ⁶ G1E-ER2 cells are added on FN-coated wells and incubated for various hours at 37 ° C. with or without SCF. Cells are then harvested and lysed at 4 ° C. for 30 minutes in lysis buffer. Cell lysates are clarified by centrifugation at 10,000 g at 4 ° C. for 30 minutes. Equal amounts of protein are fractionated on 12% polyacrylamide / sodium dodecyl sulfate (SDS) gel and electrophoretically delivered to nitrocellulose membranes. Western blot analysis is performed according to the manufacturer's instructions (New England Biolabs).

2.2.3. Cooperation between VLA-5 and c-Kit leads to enhanced and sustained FAK and MAPK (ERK-1 and ERK-2) activation

To test whether the increase in proliferation of erythrocyte progenitor cells that occur after occlusion of VLA-5 is associated with activation of downstream kinase signal transduction pathways, we have conducted the FAK and MAP-kinases (ERK-1 and ERK-). 2) activation was measured. These pathways have previously been associated with integrin-mediated signal transduction in hematopoietic cells (59). G1E-ER2 cells were deficient for 7 hours, attached to FN for various times through VLA-4 or VLA-5, lysed and subjected to Western blot analysis using anti-FAK or anti-phospho-MAPK antibodies. Let's do it. In the presence of SCF, occlusion of VLA-5 strongly induces FAK activation as early as 12 hours after stimulation and the maximum level reaches 48 hours (FIG. 3A). However, VLA-4 induced ligation also induces FAK activation at significantly reduced levels compared to activation via VLA-5 (FIG. 3A). Next, we examine the activation of MAPK (ERK) and first measure the activation of ERK in SC1 stimulated G1E-ER2 cells in suspension. Stimulation of these cells by SCF induces significant but only transient activation of ERK-1 and ERK-2 (FIG. 3B). Activation peaks 10 minutes after SCF stimulation and then declines to baseline. In contrast, in the presence of VLA-5 occlusion, SCF strongly induces ERK-1 and ERK-2 activation in erythrocytes as early as possible 10 minutes after stimulation and persists to peak levels at 120 minutes (FIG. 3C). ). However, once again, occlusion through VLA-4 results in activation of ERK-1 and ERK-2 at significantly reduced levels compared to VLA-5 (FIG. 3C). These data indicate that increased proliferation of erythrocytes associated with ligation through VLA-5 may be due in part to sustained and enhanced activation of FAK / MAP-kinase (ERK-1 and ERK-2) sequences in the cell. Present that there is.

To further investigate the association between c-Kit and / or integrin mediated proliferation of erythrocytes with the ERK pathway, we used a specific pharmacological inhibitor of MAPK (ERK) sequencing (PD98059). Factor deficient G1E-ER2 cells are cultured on CH271 or H296 or in suspension with or without SCF and PD98059. After co-culture for 48 hours, [ ³ H] thymidine is added for 6 hours and the [ ³ H] thymidine incorporation rate is measured. Despite evidence of transient ERK activation (see FIG. 3B above), PD98059 has a minimal effect on the proliferation of G1E-ER2 grown in suspension in the presence of SCF (FIG. 3D). In contrast, proliferation of G1E-ER2 cells cultured on H296 and CH271 is inhibited by 65% and 30% in the presence of PD98059, respectively. These data show that although the integrin and c-Kit stimulated growth of G1E-ER2 depends on the activation of the MAP-kinase (ERK) pathway, the extent of use of these pathways differs significantly after ligation of VLA-4 and VLA-5. Suggests.

2.2.4. Attachment via VLA-4 Induces Cell Death in G1E-ER2 Cells

SCF is an important survival factor for c-Kit positive cells and has been shown to protect certain cells from growth factor regression-induced apoptosis (29). Integrin-mediated interactions with FN have also been shown to play an important role in regulating cell survival and apoptosis in certain cells. To determine whether the decreased proliferation of G1E-ER2 cells observed in cells cultured on FN-H296 is due to partially increased cell death, we used the PI and Annexin V staining combinations to determine the presence or absence of SCF or Apoptosis in G1E-ER2 cells cultured on different FN peptides or in suspension (BSA control) in the absence was compared. G1E-ER2 cells are factor-deficient for 6-8 hours and then incubated for 48 hours on H296 or CH271 in suspension or in medium with or without SCF. Cells are harvested, stained with Annexin V and PI, and the percentage of apoptosis (annexin V + / PI −) and necrosis (annexin V + / PI +) cells are assessed using FACS assay. In experiments conducted in the absence of c-Kit stimulation, G1E-ER2 cells cultured on FN-H296 showed significantly higher cell killing rates compared to cells cultured on CH271 (52% ± 1 vs. 33.4% ±) 1, respectively, mean ± SD, p <0.05) (FIG. 4; Panel A). The viability of cells grown in suspension is similar to that observed in cultures grown on CH271 (29.3% ± 2 vs. 33.4% ± 1, respectively ± mean ± SD, p> 0.05). These data suggest that attachment to FN through LVA-4 stimulates apoptosis of G1E-ER2 cells in the absence of growth factors.

To investigate the effect of c-Kit on reversal of apoptosis, G1E-ER2 cells are cultured on FN peptides in the presence of SCF and apoptosis is measured. G1E-ER2 cells cultured on H296 in the presence of SCF show a marked improvement in survival compared to cells grown in the absence of SCF. However, rescue is only partial and significantly lower compared to cells cultured on CH271 and SCF (24.8% ± 5.3, VLA-4 vs 8.6% ± 0.2, VLA-5, mean ± SD, p <0.05) (FIG. 4; panel B). The percentage of viable cells in suspension culture in the presence of SCF is similar to that observed for cells cultured on CH271 (7.45% ± 1.45, BSA vs 8.6% ± 0.2, mean ± SD, respectively). These results suggest that the reduced proliferation observed in erythrocytes after attachment to FN via VLA-4 is due in part to an increase in the number of erythrocytes in apoptosis.

The downstream effectors of PI-3 kinase, Akt, are involved as important antagonists of apoptosis (55). Previous studies have associated reduced activation of Akt with enhanced apoptosis (55). In addition, in certain cells, expression of structurally activated forms of Akt has been shown to block apoptosis, and the use of the Akt inhibitor Wortmannin has been shown to enhance apoptosis (Refs. 60 and 61). Thus, we measured Akt activation under cell culture conditions as described above. Linkage of VLA-4 or VLA-5 induces Akt activation to different degrees in erythrocytes (FIG. 5A). VLA-4-induced Akt activation is significantly lower over the culture period as compared to VLA-5-mediated Akt activation in G1E-ER2. A 3-4 fold reduction in Akt activation via VLA-4 attachment is observed at all time points examined (FIG. 5A). To further investigate the importance of Akt activation in preventing apoptosis of these cells, we culture G1E-ER2 cells on CH271 or in suspension (BSA control) in the presence of SCF and wortmannin. The presence of wortmannin under these culture conditions leads to a significant increase in apoptosis of G1E-ER2 cells grown on CH271 (FIG. 5B). Overall, these data suggest that PI-3 kinase / Akt signal transduction cascade plays an important role in mediating erythroid apoptosis downstream from c-Kit and integrin. Attachment of G1E-ER2 cells to FN via VLA-4 significantly impairs Akt activation, and this damage may be a mechanism of enhanced apoptosis of these cells as compared to cells grown on suspension or in FN via VLA-5 have.

Discussion of Examples

In fetal development and growing animals, c-Kit and integrin-mediated signal delivery is essential for erythrocyte survival, proliferation and differentiation (1, 14, 29 and 30). Thus, erythrocyte progenitor cells provide a unique model for studying the mechanisms of c-Kit and integrin-mediated signal delivery within physiologically relevant categories. To facilitate the study of the mechanisms governing the pleiotropic response of known c-Kit and integrins in these cells, a cell line named as (G1E-ER2), similar to the primary cells of the progenitor developmental stage, is used. These cells are similar to primary erythroid progenitor cells with respect to globin and erythrocyte specific transcription factor expression and differentiation (62 and 63). In addition, G1E-ER2 cells express c-Kit, VLA-4 and VLA-5 and have been shown to attach to FN peptides at levels similar to primary erythroid progenitor cells.

The role of cell attachment to FNs during erythrocyte development has not been fully determined, but some studies have shown that FN provides a suitable fit for erythrocyte development and also provides proliferation stimulation for erythrocytes both in vitro and in vivo. As required (Refs. 32, 33, 34 and 36). The studies presented herein show significantly greater proliferation and enhanced activation of ERK in cells grown in cultures containing VLA-5 attachment sites compared to cells grown in suspensions or cultures containing VLA-4 binding sites. Prove that. In addition, our results suggest a quantitative difference in the activation of FAK and MAP-kinase ERK-1 & ERK-2 in cells grown on FN that mediate attachment through two different β1 integrin receptors. Specifically, the occlusion of VLA-4 on G1E-ER2 cells results in a significant reduction in FAK, ERK-1 and ERK-2 activation as compared to cells grown in cultures containing VLA-5 binding sites. The decrease in FAK, ERK-1 and ERK-2 activation is correlated with the reduced proliferation rate of these cells. In addition, since cells cultured on FN containing the VLA-5 binding site are more resistant to the growth-inhibitory effect of specific MEK inhibitor PD98059, the difference in activation appears to be important for cell proliferation.

In contrast, adhesion of cells via VLA-4 results in significant cell death. In addition to reduced activation of FAK and MAPK, inhibition of growth in response to linkage through VLA-4 correlates with a marked decrease in Akt activation in these cells. SCF-induced activation of c-Kit partially prevents apoptosis in these cells, but the reversal of this apoptosis is incomplete compared to cells grown on suspension or in FN mediating adhesion via VLA-5. Studies in fibroblasts have shown that integrins and growth factors can co-regulate Akt activation. In fibroblasts, activation of Akt induces inhibition of apoptosis, while in other cells activated Akt can protect cells from apoptosis in response to growth factor decline, accelerating apoptosis in these cells by predominantly-negative Akt [61]. Recently, we have demonstrated that prolonged activation of Akt by stimulation of c-Kit through membrane-bound SCF is associated with erythrocyte survival / proliferation [64]. The inventors' finding that VLA-4-mediated ligation induces reduced Akt activation and consequently induces greater apoptosis is consistent with previous studies that Akt is associated with hematopoietic cell survival. An important role for PI-3 kinase-dependent Akt activation in erythrocyte survival via integrin and / or growth factor receptor stimulation was further demonstrated in the studies presented herein by the use of the PI-3 kinase inhibitor, Wortmannin. The results suggest that the PI-3 kinase / Akt pathway is an important antagonist of cell death downstream of c-Kit and integrins in erythrocytes.

The mechanism (s) of the opposite effect of the linkage of VLA-4 and VLA-5 on the growth and survival of red blood cells is not clear. Although G1E-ER2 cells express VLA-4 and VLA-5 at comparable levels, we cannot exclude the possibility that the overall binding capacity of VLA-4 and VLA-5 to FN may be different. This difference in overall binding capacity can lead to different biological and biochemical consequences. Alternatively, the observed difference may be due to the generation of unique signals through respective occlusion of the specific α5 and α4 chains of VLA-5 and VLA-4 with FN. For example, studies presented herein using erythroid progenitors demonstrate significantly greater activation of FAK in cells grown in the presence of FN containing VLA-5 binding sites compared to VLA-4. In this regard, enhanced FAK activation via VLA-5 and c-Kit can lead to a more effective increase of Ras, thus inducing kinase sequencing downstream of Raf-1, MEK and MAPK. In fibroblasts, there is evidence of support of this model. Attachment-mediated autophosphorylation of FAK leads to increased Src, increasing tyrosine phosphorylation of FAK and subsequent binding of SH2-domain proteins including Shc and Grb2 / Sos complexes (65, 66 and 67). Formation of FAK / Src / Grb2 complexes suggests the possibility of further signal delivery to MAPK. In addition, the study suggested that overexpression of FAK caused Ras-dependent activation of MAPK [68]. As such, enhanced phosphorylation of FAK via VLA-5 and c-Kit activation will elucidate enhanced activation of ERK in cells grown on FNs that mediate adhesion via VLA-5. In contrast, recent studies using primary human periodontal ligament fibroblasts have shown that adhesion to FN through VLA-4 causes reduced FAK expression. Changes in FAK expression after attachment via VLA-4 in these cells are suggested to be due to the activation of caspase cascades (69). As such, our data in addition to the above published results provide a mechanism by which the adverse effects of integrin ligation can be mediated in red blood cells.

Previous studies using long-term human bone marrow cultures have demonstrated that hematopoietic cell proliferation is inhibited in response to direct attachment to bone marrow epilepsy via integrin receptors (17). The data presented herein suggest that VLA-4 mediated ligation induces a distinctive signal in erythroid progenitors as compared to VLA-5 mediated ligation to FN. The result of these signals is varying mortality versus proliferation. Taken together, these data support the view that specific integrin-FN interactions in hematopoietic cells can regulate cell viability and proliferation, and these effects can be regulated through ligand-induced stimulation of growth factor receptors. .

The invention is illustrated and described in detail in the above description, including specific examples, which are illustrative, but are not to be considered limiting in nature, and merely illustrate, describe, and describe preferred embodiments, all of which are included within the scope of the invention. It is understood that changes and modifications are intended to be protected.

Reference

The following references, and all other references cited herein, are indicative of the levels of those skilled in the art, each of which is incorporated herein by reference in its entirety as if each document were individually cited and cited individually in its entirety.

Claims (25)

Hematopoietic cell populations containing erythrocyte progenitor cells are cultured under conditions effective to enhance proliferation of erythrocyte progenitor cells in the presence of a polypeptide having a VLA-5 binding site, thereby attaching the erythrocyte progenitor cells to the VLA-5 binding site during culture. A method of enhancing the proliferation of erythrocyte progenitor cells, including treatment. The method of claim 1, wherein the polypeptide is a fibronectin fragment. The method of claim 2, wherein the polypeptide is a recombinant. 4. The method of claim 1, wherein at least about 60% of the hematopoietic cell population consists of erythrocyte progenitor cells. The method according to any one of claims 1 to 4, wherein the culturing is carried out in the presence of hepatocellular factor. 6. The method of claim 1, wherein the hematopoietic cell population is a human cell population. 7. The method of claim 1, wherein the culturing is carried out for a period of time and under conditions sufficient to multiply the number of erythroid progenitor cells by at least about one fold. 8. The method of claim 7, wherein the culturing is carried out for a period of time and under conditions sufficient to multiply the number of erythroid progenitor cells by at least about two times. The method of claim 1, wherein at least about 80% of the hematopoietic cell population consists of erythrocyte progenitor cells. 10. The method of any one of claims 1-9, wherein the VLA-5 binding site is the only cell binding site on the polypeptide. Hematopoietic cell populations containing erythrocyte progenitor cells were cultured under conditions effective to induce apoptosis of erythroid progenitor cells in the presence of a polypeptide having a VLA-4 binding site, thereby erythroid progenitor cells were subjected to the VLA-4 binding. A method of inducing apoptosis of erythroid progenitor cells, including attaching to a site. The method of claim 11, wherein the polypeptide is a fibronectin fragment. The method of claim 12, wherein the polypeptide is a recombinant. The method of any one of claims 11-13, wherein at least about 60% of the hematopoietic cell population consists of erythrocyte progenitor cells. The method according to any one of claims 11 to 14, wherein the culturing is carried out in the absence of hepatocellular factor. The method of claim 11, wherein the hematopoietic cell population is a human cell population. The method of claim 11, wherein the culturing is carried out for a period of time and under conditions sufficient to cause apoptosis of at least about 10% of erythroid progenitor cells in the cell population. The method of claim 17, wherein the culturing is carried out for a period of time and under conditions sufficient to induce apoptosis of at least about 20% of erythroid progenitor cells in the cell population. 19. The method of any one of claims 11-18, wherein at least about 80% of the hematopoietic cell population consists of erythrocyte progenitor cells. The method of any one of claims 11-19, wherein the VLA-4 binding site is the only cell binding site on the polypeptide. A method of treating a patient, comprising administering to the patient a cell population prepared by the method of any one of claims 1 to 20. A medium for culturing hematopoietic cells for enhancing proliferation of erythrocyte progenitor cells, the medium comprising a polypeptide having a VLA-5 binding site. A cell composition comprising proliferated erythrocyte progenitor cells obtained by the method according to any one of claims 1 to 10. A medium for hematopoietic cell culture inducing apoptosis of erythroid progenitor cells, the medium comprising a polypeptide having a VLA-4 binding site. Cell composition obtained by the method according to any one of claims 11 to 20.