WO2006101444A1

WO2006101444A1 - Improved proliferative capacity using cell-cell fusion generated hybrids

Info

Publication number: WO2006101444A1
Application number: PCT/SE2006/000363
Authority: WO
Inventors: Quamrul Islam
Original assignee: Quamrul Islam
Priority date: 2005-03-22
Filing date: 2006-03-22
Publication date: 2006-09-28
Also published as: CA2602136A1; EP1866411A1

Abstract

The current invention concerns improving cell proliferative potential and prolonging the expected lifespan of differentiated, as well as non-differentiated cells. This is achieved through the generation of activated cells which in turn stimulate the growth of cells displaying non-dividing/senescent cell phenotypes. Activated cells are induced using cells or derivatives of the F7 cell line, and may be intra- and/or inter-species cell hybrids generated by cell-cell fusion or can be achieved using conditioned media from F7 cell culturing. Resulting activated cells have superior proliferation potential compared to the non-activated parental cell types and can be characterised by chromosomal heteroploidy/aneuploidy . The improved growth properties of the activated cells generated can be utilized directly in facilitating the clinical and medical treatment of diseases in humans and other mammals and for the repair of damaged tissues/organs, or can be utilized indirectly to provide media and/or factors with unique growth promoting and/or sustaining properties. Generation of hybrid or activated cells in accordance with the present invention provide an alternative to conventional embryonic and adult stem cells, and circumvent the need for the specialised and costly cell culture techniques required in current stem cell applications.

Description

TITLE :

Improved proliferative capacity using cell-cell fusion generated hybrids

BACKGROUND OF THE INVENTION:

In contrast to most transformed and tumour cells, normal somatic cells undergo a limited number of divisions in culture before entering a non-dividing state called replicative or cellular senescence (Hayflick, L., 1965 "The limited in vitro lifetime of human diploid cell strains" Exp Cell Res 37:614-636). After cessation of division, senescent cells do not die immediately, but remain viable for extended periods of time if adequate nutrient is supplied through regular renewal of medium in a carefully maintained culturing environment. Senescent cells are, however, unable to synthesize DNA in response to serum stimuli (Goldstein, S., 1990 "Replicative senescence: the human fibroblast comes of age" Science 249:1129-33) and can be distinguished by a characteristic enlarged, flattened morphology.

Several types of senescence have been described in the literature (Ben-Porath I., & Weinberg R. A., 2005 "The signals and pathways activating cellular senescence" Int J Biochem Cell Biol 37:961-976; Lloyd A. C, 2002 "Limits to lifespan" Nat Cell Biol 4:E25-E27; Serrano M., & Blasco M.A., 2001 "Putting the stress on senescence" Curr Opin Cell Biol 13:748-753; Sherr CJ. , & DePinho R.A. , 2000 "Cellular senescence: mitotic clock or culture shock?" Cell 102:407- 410) . These include;

- replicative senescence (associated with successive telomere shortening) ,

- premature or induced senescence (associated with oncogenic activation or introduction of activated oncogenes into normal cells) ,

- stress-induced senescence (resulting from inadequate culturing conditions) . It has been theorised that replicative senescence in^' normal cells is a natural safeguard against unrestrained cell proliferation and helps to prevent neoplastic transformation, as the acquisition of unlimited proliferative potential is considered to be a critical step during tumour development.

Over the last two decades, scientists have recognised the benefits to be gained in terms of being able to treat disease and regenerate damaged tissue and organs if the limited replicative potential of a variety of cell types could be overcome. Efforts to solve cell proliferation problems have previously focused on stem cells, which have the characteristic features of being able to renew themselves and have the capacity for differentiation into cells from different lineages, if specific differentiation stimuli are provided. Different stem cell populations, however, have demonstrated variation in potential and are now known to range from totipotent (ability to form into either embryo or placental cells) , to pluripotent (ability to form into almost all germ layer cells), to multipotent (ability to form into a limited range of cells from various lineages) to unipotent (ability to generate one cell type, often with limited growth potential) .

Three types of embryo-derived stem cells have been extensively studied and have been used both as models for elucidating cell lineage mechanisms and regulation of gene expression during mammalian embryogenesis, as well as providing a vehicle for introducing genomic modification:

(i) embryonic stem (ES) cells derived from the inner cell mass of developing blastocysts which are pluripotent and able to differentiate into cellular derivatives representative of all three primary germ layers (ii) embryonic germ (EG) cells which are derived from the primordial germ cells of 5- to 10-week fetal gonadal ridge and (iii) embryonal carcinoma (EC) cells that develop from testicular tumors (teratomas and teratocarcinomas) . These EC cell lines provide a useful model system for many applications as they are easier to grow and maintain than ES and EG cell lines and share many features with ES cells, but the range of cell types exhibited by differentiated EC cells is less than that shown by ES cells because they are chromosomally abnormal and consequently have limited clinical potential.

Ethical issues regarding the use of embryonic stem cells for therapeutic cloning, as well as instability in maintaining and difficulties in directing cell-type specific differentiation have subsequently resulted in a shift of research focus to the manipulations of adult stem (AS) cell populations.

It has recently been discovered that in adults, most of the major organs of the body harbour populations of stem cells which function to rescue and repair damaged tissue, including bone marrow, muscle, heart, skin, intestine, liver, lung, prostate, central nervous system, mammary gland, and others. Similar to ES cells, however, AS cells can lack tissue-specific characteristics and require the influence of appropriate signals to differentiate into specialized cells. These cells may be the same as, or distinct from, that of the precursor. Recent reports suggest that tissue-specific stem cells are in fact capable of a much wider spectrum of differentiation than was initially thought, as these were previously believed only to be able to develop into cells of related lineages.

Despite certain limitations such as developmental inefficiencies and lack of long-term self-renewals, AS cells are currently under investigation for use in various genetic manipulations and for treating human diseases through cell-based therapies. For such utilities, some investigators have even carried out cell fusion experiments, using somatic cells and embryonic stem cells in order to obtain reprogrammed cells which display characteristics of the differentiated somatic cell, but which also display a degree of the improved self-renewal and developmental capacity of the stem cell. These resulting hybrids often retain a near-diploid chromosome complement (Matveeva N.M., et al, 1998 "In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes". MoI Reprod Dev 50:128-38; Serov 0., et al, 2001 "Embryonic hybrid cells: a powerful tool for studying pluripotency and reprogramming of the differentiated cell chromosomes" An Acad Bras Cienc 73:561-8; Medvinsky A., & Smith A., 2003 "Stem cells: Fusion brings down barriers". Nature 422:823-5). Due to the drastic loss of chromosomes which occurs from each cell parent, however, these hybrids are expected to suffer from limitations in their expression of cellular factors necessary for generative purposes.

Other attempts have therefore been made to generate differentiated cells with indefinite, or much increased proliferative potential. These include the use of retrovirus introduction, such as SV40 transformation, to "immortalise" cells, as well as attempts to focus on a defined number of genetic alterations in normal cells, such as inactivation of the Rb/pl6 pathway, simultaneous disruption of p53 and Rb pathways and the activation of the TERT enzyme (Telomerase Reverse Transcriptase) . No consensus has been reached, however, on the number of genetic events required for the immortalization of a normal cell (Hahn W. C. et al. 2002 "Enumeration of the simian virus 40 early region elements necessary for human cell transformation" MoI Cell Biol 22:2111-23). Nevertheless, by forced expression of the catalytic component of telomerase (TERT) , either alone or in combination with oncogenes it is often possible to generate cell lines with indefinite growth potential from normal cells (Drayton S., & Peters G., 2002 "Immortalisation and transformation revisited" Curr Opin Genet Dev 12:98-104; Newbold R. F., 2002 "The significance of telomerase activation and cellular immortalization in human cancer" Mutagenesis 17:539-50; Harley CB. , 2002 "Telomerase is not an oncogene" Oncogene 21:494-502) . It should be emphasized that normal human cells are more resistant to immortalization than the cells of other mammalian species, particularly cells derived from many rodent species spontaneously immortalized in culture (Wright W. E., & Shay J. W., 2000 "Telomere dynamics in cancer progression and prevention: fundamental differences in human and mouse telomere biology" Nat Med 6:849-51; Artandi S. E., & DePinho R.A., 2000 "Mice without telomerase: what can they teach us about human cancer?" Nat Med 6:852-5; Akagi T., Sasai K., & Hanafusa H., 2003 "Refractory nature of normal human diploid fibroblasts with respect to oncogene- mediated transformation" Proc Natl Acad Sci U.S. A 100:13567-72; Simonsen J. L., et al, 2002 "Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells" Nat Biotechnol 20:592-6; Veitonmaki N., et al, 2003 "Immortalization of bovine capillary endothelial cells by hTERT alone involves inactivation of endogenous pl6lNK4A/pRb" FASEB J, 17:764-6).

Early somatic cell genetic studies involving the fusion of normal human fibroblasts with tumourigenic immortal cell lines indicated that cellular immortality (defined as the capacity of indefinite proliferation of cells in culture) and cellular tumourigenicity (defined as tumour forming ability of cells in syngenic or immuno-compromised mice) are dominant traits (Croce CM., & Koprowski H., 1974 "Positive control of transformed phenotype in hybrids between SV40-transformed and normal human cells" Science 184:1288-1289; Klinger H. P., 1980 "Suppression of tumorigenicity in somatic cell hybrids. I. Suppression and reexpression of tumorigenicity in diploid human x D98AH2 hybrids and independent segregation of tumorigenicity from other cell phenotypes" Cytogenet Cell Genet 27:254-66). Subsequent cell fusion studies in conjunction with cytogenetic analyses, however, revealed that the lack of suppression of tumourigenicity in the early studies was due to massive loss of chromosomes originating from the normal fibroblast parent cell. The results of later studies led to the conclusion that tumourigenicity is a recessive trait and that the inactivation (loss of function) of both alleles of the tumour suppressor gene(s) is required for the expression of tumorigenic phenotype (Harris H. 1993 "How tumour suppressor genes were discovered" FASEB J 7:978-9). Recent studies, however, have provided evidence that aneuploid cells, which are the result of spontaneous fusions between two types of cells co-cultured in vitro, can be transplanted, at least in mouse models, and form differentiated cell-types without tumours being formed (Que J., et al, 2004 "Generation of hybrid cell lines with endothelial potential from spontaneous fusion of adult bone marrow cells with embryonic fibroblast feeder" Jn Vitro Cell Dev Biol Anim 40:143-149).

In addition to the tumourigenicity studies described above, it has previously been claimed that the phenotype of cellular senescence is dominant over the phenotype of cellular immortality (Pereira-Smith O.M., & Smith J. R., 1983 "Evidence for the recessive nature of cellular immortality" Science 221:964-6; Pereira-Smith O.M., & Smith J. R., 1988 "Genetic analysis of indefinite division in human cells: identification of four complementation groups" Proc Natl Acad Sci U. S. A 85:6042-6). Experimental evidence from numerous recent and old studies, however, involving the fusion of diverse immortal cells including the ES (embryonic stem) , EG (embryonal germ) , and EC (embryonal carcinoma) cell lines with different types of normal cells suggests that hybrid cells can be generated with unlimited proliferative capacity (Takagi N., 1997 "Mouse embryonal carcinoma cell-somatic cell hybrids as experimental tools for the study of cell differentiation and X chromosome activity" Cancer Genet Cytogent 93:48-55). These studies indicate that the fusion of immortal cells with normal cells does not always produce hybrid cells with a limited proliferative potential, as previously expected. It has been argued that the escape of senescence in these hybrids is due to rapid loss of large numbers of chromosomes derived from normal cells, similar to the situation observed in the tumourigenicity studies (Ran Q., & Pereira-Smith O.M., 2000 "Genetic approaches to the study of replicative senescence" Exp Gerontol 35:7-13) .

SUMMARY OF THE INVENTION:

The current invention concerns improving cell proliferative potential and prolonging the expected lifespan of differentiated, as well as non-differentiated cells. This is achieved through the generation of activated cells which in turn stimulate the growth of cells displaying non-dividing/senescent cell phenotypes . Activated cells are induced using cells or derivatives of sub-populations of cells derived from the GMO5267 cell line, and may be intra- and/or inter-species cell hybrids generated by cell- cell fusion or can be achieved using conditioned media from cell culturing of sub-populations of cells derived from the GMO5267 cell line. Resulting activated cells have superior proliferation potential compared to the non-activated parental cell types and can be characterised by chromosomal heteroploidy/aneuploidy. The improved growth properties of the activated cells generated can be utilized directly in facilitating the clinical and medical treatment of diseases in humans and other mammals and for the repair of damaged tissues/organs, or can be utilized indirectly to provide media and/or factors with unique growth promoting and/or sustaining properties. Generation of hybrid or activated cells in accordance with the present invention provide an alternative to conventional embryonic and adult stem cells, and circumvent the need for the specialised and costly cell culture techniques required in current stem cell applications.

DETAILED DESCRIPTION OF THE INVENTION:

By conducting multiple experiments with high reproducibility and subsequent cytogenetic analyses of the cells and hybrid cells formed from fusions involving a variety of cell types including senescent cells, we have found unequivocally that the presence of two nearly complete diploid sets of chromosomes (4N) derived from normal cell types (for illustration purposes only, fibroblasts are described in the Examples section below) actually enhances the proliferation of hybrid cells, instead of blocking cell division in accordance with currently accepted dogma. Our results clearly demonstrate that despite the presence of a tetraploid (or near tetraploid) genome derived from normal and/or senescent parental cells, resultant hybrid cells can proliferate indefinitely. Our results question the validity of the hypothesis of the dominant nature of the phenotype of cellular senescence and provide, in one embodiment of the invention, the potential for reprogramming even the genome of senescent cells efficiently by cell fusions involving cells comprising a tetraploid or near tetraploid chromosomal complement and immortal and/or moderately proliferative cell phenotypes .

The present invention comprises: the generation of novel activated cells including intra-specific as well as inter-specific cell hybrids, utilising cell fusion techniques, for the purpose of generating hybrid cells comprising particular karyotypes characterised by chromosomal aneuploidy/heteroploidy and, in a further embodiment of the invention, the direct use of these cell- hybrids for the treatment of disease, the regeneration of tissues and repair of tissues and organs following intrinsic or extrinsic damage; the generation and use of cells or cell-hybrid fusions for autologous (cells from the same subject/patient) or allogenic transplantations; the generation and use of cells such as hybrids in order to obtain growth media for direct and indirect use in providing growth factors for tissue-cell maintenance prior to transplantations, for use in reactivation and extension of the normal lifespan of senescent and/or growth limited cell-types and for the treatment of disease, the regeneration of tissues and repair of tissues and organs following intrinsic or extrinsic damage.

In a preferred embodiment of the invention, intra-specific hyper-tetraploid and hypo- and/or hyper-hexaploid hybrids are generated, or spontaneously derived near-tetraploid normal cells selected, in initial phases of the present invention (in contrast to conventional cell fusion procedures where 4N cells are generally the end-product) . Two parental cell types are co-cultured under appropriate conditions for varying lengths of time and subsequently induced to undergo cell fusion using chemical agents, normally polyethylene glycol (PEG) . Cell fusion is then followed by antibiotic selection in order to ensure the removal (death) of parental cell-types and ultimately yield the heteroploid hybrids of this invention, which display significantly enhanced growth potential compared to the parental cell-types. In a further preferred embodiment of the invention,

"activator cells" derived from sub-populations of cells derived from the GMO5267 cell line, described in Example 3 below, are used directly to activate the non-dividing senescent cells to proliferate. In the stimulation of senescent cells into actively dividing phenotypes (activated cells) , co-culturing with cells derived from the sub-populations of the GMO5267 cell line may be employed, with subsequent removal of "activator" cells, for example by antibody or chemical selection or by cell sorting. In another embodiment of the invention, conditioned media obtained from the cultured sub-populations of cells derived from GMO5267 cells, and their decendants can be directly employed for the conversion (or reprogramming) of non-activated cells, which are not in actively dividing phases of the cell cycle, into activated forms.

An important novel characteristic of the present invention compared to previous cell fusion manipulations for therapeutic utilities, is that reprogramming of participating cell-types, if required, can be carried out prior to cell fusion. This can be achieved, for example, using cell culture media or purified factors originating from the media of existing cell hybrids, as well as parental cells, using differentiation stimuli known and previously described within the art .

Cell fusions can also be carried out in order to achieve reprogramming of cells. In the context of the present invention, reprogramming utilising cell fusion may be of particular therapeutic use in tissue/organ repair procedures where autologous cells and subsequent reintroduction and/or transplantation of allogenic and/or autologous cells for the purpose of repair of damaged tissues and organs is carried out. Using cell fusion to generate heteroploid cell hybrids in accordance with the present invention allows even senescent cells from an individual to be reprogrammed and rejuvenated for use in adoptive autologous or allogenic therapeutic disease treatment regimes and for the repair of damaged tissue and organs . In one embodiment of the invention, the cell-hybrids of the invention possess the ability to be maintained in stasis, in resting or dormant growth states, when nutritional conditions are limited. This embodiment of the invention is exemplified in Example 7, below, describing mouse-mouse hybrids. The proliferation of such static hybrid cells can subsequently be reinitiated by trypsinization of the cells and subsequent low density plating in fresh culture media. Characteristically, some hybrid cells of the invention subsequently even display enhanced propagative properties compared to the growth characteristics of the original (parental cell) populations from which the hybrid cells are derived.

In another embodiment of the invention, hybrid cells are generated which are characterised by their ability to continuously propagate in culture conditions where nutrition is not restricted. This embodiment of the invention is exemplified by the pig-mouse hybrid cell system described in Examples 4, 5 and 6, below.

As outlined above, yet another embodiment of the invention relates to the recovery of media which has been previously used for the growth and/or maintenance of the cell-hybrids of the invention. This media, comprising factors secreted by the cell-hybrids, can be directly or indirectly used to restore proliferative status to related or unrelated cells which have undergone senescence or have reached a retarded proliferative state. It will be readily understood by those skilled in the art that a variety of methods may be employed to isolate and purify factors secreted into culture media by the hybrid cells. The purification and use of isolated and purified factors secreted by the hybrid cells of the invention for the purpose of restoring proliferative status to cells which have undergone senescence is also envisioned to lie within the scope of the present invention.

Despite certain limitations such as developmental inefficiencies and lack of long-term self-renewals adult stem cells are currently under investigation in genetic manipulations for their usefulness for therapeutic purposes. We have generated cell hybrids in vitro by fusing adult stem cells with differentiated cells to establish that the experimentally induced cell hybrids express similar properties to stem cells. We believe that establishment of hybrid cell systems in vitro with properties which mimic the characteristics of stem cells can provide a better way of treating many human diseases by local application of pure populations of hybrid cells with a substantially more efficient regenerative capacity. The hybrid cells used for the treatment of individual patients can be derived from the individual patient in need of treatment, so circumventing adverse autoimmune responses.

The following examples are included for the purpose of further illustrating the invention and are not intended to limit the scope of the invention in any way.

EXAMPLES :

Example 1. Increased frequency of hybrid formation by optimization of cell fusion conditions

A number of conventional polyethylene glycol (PEG) cell fusion techniques are known in the art and can be used for the generation of hybrid cells. We have normally used at least one monolayer parental cell-type in all cell fusion experiments in order to increase the frequency of hybrid formation (see for example Islam, K. & Islam M. Q., 1994 "Assignment of TKl encoding thymidine kinase to Syrian hamster chromosome 9 by microcell-mediated chromosome transfer" Cytogenet Cell Genet 66:177-180; Islam M. Q. & Islam K., 2000 "Suppressor genes for malignant and anchorage- independent phenotypes located on human chromosome 9 have no dosage effects" Cytogenet Cell Genet 88:103-9). Suitable culture media, duration of trypsin treatment, ratio of the two parental cell-types in co-culture, duration of co-culture before PEG treatment and evaluation of the condition of fused cells after PEG treatment must all be taken into consideration when optimising the cell fusion procedure in order to achieve high frequency hybrid formation. Duration times for each stage of the procedure, choice of culture media etc is dependant on the types of cells to be fused.

In the generation of hybrid cells of this invention, the following exceptions to conventional procedures are used; a) greatly reduced incubation times of co-cultured cells prior to fusion, b) varying the amounts of phytohemagglutinin (PHA) , compared to the accepted protocols known in the art, that are added prior to PEG exposure, c) regular monitoring of PEG-treated cells (by incubating at 37°C at least for three hours to ascertain that cells are not affected adversely because of PEG treatment) . If the fused cells showed drastic changes in morphology, they were washed again with serum free medium in order to reduce the toxic effects of PEG. Optimisation of these parameters results in substantially increased frequencies of hybrid cell formation.

In brief, after individual trypsinisation of two types of cells, cells are mixed in a centrifuge tube, the mixed cells plated in a 6-cm culture dish and then incubated for three to four hours to allow the attachment and spreading of cells so that cell-cell contact can take place before inducing fusion. Twenty to thirty minutes prior to PEG treatment, 100 μg/ml PHA is added to the medium of culture dish. Cells are PEG treated for 60 seconds (45% PEG) and then washed using several changes of serum free culture media. 24-48 hours post fusion selection procedures are then applied (as described above) and subsequently hybrid cells are selected in appropriate medium, depending on the cell types to be fused.

Example 2. Generation of cell-lines with selectable markers: GM05267-Neo^R

GMO5267 is a SV40-transformed immortal mouse cell line, deficient for the enzyme hypoxanthine-phosphoribosyl-transferase (HPRT) . The GMO5267 cells were obtained from the National Institutes of General Medical Sciences (NIGMS) , Camden, NJ, USA. These cells,

11 of fibroblast origin, derive from mouse kidney and come from a cell line originally named GM5267 that was established in order to study the testicular feminisation locus (Migeon B. R. efc al, 1981 "Studies of the locus for androgen receptor: localization on the human X chromosome and evidence for homology with the Tfm locus in the mouse" PNAS USA, 78:6339-6343).

GMO5267 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 15% fetal calf serum, 1% DMEM nonessential amino acids, and 1% penicillin-streptomycin (reagents obtained from Invitrogen Life Technologies) . A G418- resistance gene was introduced into the mouse parental GMO5267 cell line by a modified calcium-phosphate co-precipitation method as follows :

Exponentially growing cells were trypsinized, seeded into 6-cm culture dishes and incubated overnight at 37°C in a 5% CO₂ atmosphere. 20 μg of plasmid DNA (pSV2neo) was mixed with 0.5 ml of 0.25 M CaCl₂ and 0.5 ml of 2 x BES-buffered saline was added. This calcium phosphate-DNA mixture was kept for 20 minutes at room temperature before adding 1 ml drop-wise to the plated cells, mixing gently and allowing to stand for 30 min at room temperature prior to further overnight incubation (as described above) . The medium was subsequently removed and transformed cells selected with fresh DMEM medium containing G418. Cells were maintained in culture with twice weekly media replenishment. After three weeks, a single colony of G418-resistant cells was transferred to a culture flask for further multiplication. This cell clone was designated GM05267-Neo^R.

Example 3. Isolation of subpopulations of the Cell Line GMO5267

GM05267-Neo^R cells were grown and sub-cultured for several passages in appropriate culture media with concomitant observation of their growth properties. Sub-cloning and subsequent selective rounds of culturing gave rise to a number of cell lines, which displayed unique growth characteristics. These selective culturing procedures allowed stable sub-lines to be established. These cells display substantially improved growth properties compared to the parental cell type.

Example 4. Generation of inter-specific pig-mouse cell hybrids with improved generative properties compared to parental cell types .

Cells of the GMO5267-Neo^R mouse cell clone (HAT-sensitive and G418-resistant) , described in Example 2, above, or cells originating from selective sub-culturing, such as those described in Example 3, above, and normal pig fibroblast cells of the strain AG08114 (HAT-resistant and G418-sensitive; NIA, Aging Cell Culture Repository, Coriell Institute for Medical Research, Camden, NJ, USA) were trypsinized separately and equal numbers of mouse cells and pig fibroblast cells were mixed in centrifuge tubes and seeded into 6-cm tissue culture dishes. After 3-4 hours, cell fusion was induced by- adding 2 ml of polyethylene glycol (PEG-Sigma, MW 1,500) prepared in serum-free DMEM, (45% w/v) , for 1 min. The cell layer was subsequently washed with serum-free DMEM and cells incubated overnight (see above) . The cells were then trypsinized, suspended in medium containing G418 plus HAT, and plated into 10-cm culture dishes. After 7-10 days, hybrid colonies were individually trypsinized, using cloning rings and transferred into culture flasks. Pig-mouse hybrid cell lines derived from the neomycin- resistant mouse cell line GM05267-Neo^R were designated, in series, with the prefix PMN. A total of 19 independent PMN-hybrid cell lines were generated.

Chromosomes of the parental cell lines/strains were identified and counted from printed images of the metaphases from 20 to 35 metaphase cells from each cell strain/line using a CCD camera and the CytoVision software program (Applied Imaging) . Chromosome preparation and chromosome banding procedures were as described previously (Islam M. Q. & Levan G., 1987 "A new fixation procedure for quality G-bands in routine cytogenetic work" Hereditas 107:127- 30; Islam M. Q., et al, 1995 "Monochromosome transfers to Syrian hamster BHK cells via microcell fusion provide functional evidence for suppressor genes on human chromosome 9 both for anchorage independence and for tumorigenicity" Genes Chromosomes Cancer. 13:115-25). Hybrid cell chromosomes from each parental species were counted separately and the total number of chromosomes determined by adding the numbers from the two species. In selected cases, chromosomes were analyzed by manual karyotyping using the CytoVision program.

Using similar procedures to those described above,

GM05267-Neo^R cells, or its sub-lines, were fused with cells from the pig fibroblast strain AG12077 (NIA, Aging Cell Culture Repository, Coriell Institute for Medical Research, Camden, NJ, USA) . Following polyethylene glycol fusion and several days of culture selection, a total of 15 hybrid fusion cell lines were obtained.

Table 1 Chromosome composition of the pig-mouse cell hybrid-PMN series containing near-tetraploid pig and hyper-haploid mouse chromosomes

Table 1 shows the results of cytogenetic analyses of a number of randomly selected hybrid cell-lines from the above two examples. No hybrids generated using GM05267-Neo^R cells displayed a systematic loss of pig chromosomes after karyotyping. These hybrids were seen to retain approximately 4 sets (4N) of pig chromosomes. In contrast, loss of mouse parental chromosomes from hypo-diploid chromosome numbers was observed in the individual PMN-series hybrid cell-lines. Mouse chromosome loss was found to be non-random, with at least one copy of each mouse chromosome retained in the hybrid cells .

Example 5 : PMN hybrids have selective growth advantage over normal pig fibroblasts

To determine if the PMN hybrids containing nearly one and half sets of mouse and four sets of pig chromosomes have selective growth advantage over the pig parental fibroblast cells, we fused the GM052β7-Neo^R mouse cells with the fibroblasts of pig and the hybrids were selected in HAT medium. This so-called "half-selection" would allow the pig parental fibroblast cells to survive in the HAT selective medium and grow side by side with the hybrid cells. After HAT selection, the resultant mixture of hybrids and pig parental cells were subjected to cytogenetic analysis after two rounds of passaging. The results of these analyses are presented in Table 2.

Table 2 The selective growth advantage of the pig-mouse hybrids (PMN) , containing less than 2N mouse and near-4N pig chromosome complements, over the hybrid cells containing other types of genomic chromosomal combinations. The table also shows the growth of the pig parental fibroblasts, measured by the frequency of metaphase cells present in the chromosome preparations of competitively growing mixed cell cultures derived from innumerable cell colonies.

^a Derived from a common cell fusion experiment but fused cells were divided equally 24h post-fusion and allowed to grow in two different selective media (in order to make an internal control of the fusion experiment) . ^b Hybrid cells were selected in HAT+G418 in order to eliminate both mouse and pig parental cells and allow the growth of hybrid cells only (served as a control) . All hybrid cell colonies of the fusion were pooled into a single mass culture and chromosome preparation was made at passage two. ^c Fused cells were selected in HAT medium in order to eliminate only the mouse parental cells but not the pig parental fibroblasts and the hybrid cells. All HAT-resistant cell colonies were pooled and chromosome preparation was made from the presumable mixture of cells containing hybrids as well as the pig parental fibroblast cells at passage two.

It could be seen that most of the hybrid cells (94% of the metaphases) contained nearly one and a half sets of mouse and four sets of pig chromosomes. The control experiment showed comparable frequencies of hybrid metaphase cells (93%) with similar genomic constitutions (Table 2) . The majority of the cells of the original GMO5267 cell line purchased from the NIGMS possessed similar karyotypes to the subpopulations of GMO5267 derived cells. The major differences observed is characterised by the presence two copies of chromosome 6 in the original GMO5267 cell line and single copy of chromosome 6 in the GMO5267-derived cells. We therefore fused the GMO5267 cell line (G418-sensitive) with normal pig fibroblasts in order to examine growth properties. Hybrids were grown in HAT medium in order to allow the pig parental cells grow side by side with the hybrids to test whether the hybrid cells or the pig parental cells are fast growing. Cytogenetic analysis of the resultant mixed cells of hybrids and pig fibroblasts at passage three showed metaphases of mostly hybrid cells. These contained 4N pig genomes together with hypo-diploid mouse genome. Chromosome analysis after three additional passages showed no metaphases of pig fibroblast cells. Example 6. Generation of inter-specific pig-mouse hybrids by fusing pig mesenchymal stem cells with GMO5267-derived aneuploid cells

Pig mesenchymal stem cells (PMSCs), described previously (Ringe J. et al, 2002 "Porcine mesenchymal stem cells. Induction of distinct mesenchymal cell lineages" Cell Tissue Res, 307:321-7), were fused with aneuploid subpopulatins of cells derived from GMO5267. Hybrids were selected in medium containing HAT (in order to eliminate non-fused GMO5267-derived cells) and G418 (in order to eliminate non-fused PMSCs) . A total of 16 randomly selected hybrids were subjected to cytogenetic analysis and the results of these analyses are shown in Table 3.

Table 3 The chromosome composition of inter-specific pig-mouse hybrids generated by fusion of PMSC with a GMO5267-derived mouse cell line

These results indicate that the pig-mouse hybrids containing nearly four sets (4N) of normal pig chromosomes and less than two sets (2N) of mouse chromosomes have selective growth advantage over hybrid cells with other genomic constitutions, including pig parental fibroblasts. Growth of these hybrids could be further enhanced by frequently changing the culture medium.

Hybrids were subsequently grown, cryopreseved, recovered and some have undergone more than 120 population doublings without loss of proliferative properties. In contrast, the parental fibroblasts senesced after only a few passages in cell culture.

The fusion experiments described above, of a hypo-diploid mouse cell line with normal pig diploid fibroblasts results in hybrids (PMN and PMSC hybrids) containing tetraploid pig genome/chromosomal complements (4N) along with hypo-diploid mouse chromosomal complements (2:1 fusion). Fusions have been repeated several times with the same result i.e. the hybrids always contain 4N pig chromosomes and hypo-diploid mouse chromosomes (both in clonally isolated hybrids and mass cultures) . This result contradicts the widely accepted view that hybrid cells containing 2N chromosomes from the normal parent should not grow for long in culture because of the dominant nature of limited proliferation of the normal cells. However, the PMN and PMSC hybrids with overloaded normal pig chromosomes (4N) not only divide indefinitely in culture but they grow faster than normal pig fibroblasts, as evident from co-culturing of hybrids and pig parental fibroblasts (50:50) . G- banded analysis has revealed that PMN and PMSC hybrids carry morphologically normal pig chromosomes without any structural abnormality. It appears that, whatever chromosome number may come from the nuclei of the normal cell parent, fusion with the immortal cell will erase the original genetic program of the normal-senescent cells and convert them into an immortal cell program by nuclear reprogramming. Thus immortality behaves as a dominant trait.

Example 7. Generation of intra-specific mouse cell hybrids .

A series of five intra-specific mouse cell hybrids were created: two series of hybrids were generated by fusing adult mouse stem cells (LTC-4D cell line) either with normal non-transformed mouse fibroblasts T(4;X)37H or with cells of transformed murine GMO5267-derived cells: (a) fusion of mouse LTC-4D with mouse T(4;X)37H cell line resulted the LTC-4D-T37 hybrids; (b) fusion of mouse LTC-4D with mouse GMO5267-derived cells resulted the LTC-4D- GMO5267-derived hybrids.

(a) The LTC-4D cell line was isolated from the mouse strains, C57B1/6 (IDUA-KO) as described in Meirelles Lda, S. & Nardi, N. B., 2003 "Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion and characterization" Br J Haematol' 123:702-711). The IDUA-KO mouse strain is deficient for alpha-L- iduronidase gene and the LTC-4D cell line was derived from a male mouse carrying the NeoR gene in the genome. Characterization of the established LTC-4D cell line using a battery of in vitro assays has previously determined that these cells have multi-lineage differentiation capabilities. In order to discriminate the parental origin of chromosomes in subsequent mouse intra-specific cell hybrids, primary mouse fibroblasts derived from the mouse strain T(4;X)37H, carrying reciprocal translocations involving chromosome 4 and X, were chosen for fusion with the LTC-4D cell line. The use of the T(4;X)37H fibroblast cells in fusions allows the genomic contribution of parental cells in the resultant hybrids to be determined (1:1 or 1:2 or 2:1 hybrids) by counting the copy numbers of the t(4;X) marker chromosome and normal X, and Y chromosome. A Hygromycin-resistant cell line HygR T(4;X) had been previously generated by retroviral infection of the T(4;X)37H fibroblasts.

HygR T(4;X) fibroblasts were fused with the LTC-4D cell line from C57B1/6 (IDuA-KO) according to the procedures outlined in Example 1, above. After fusion, hybrid cells were selected in DMEM containing G418 (in order to eliminate T(4;X)37H cells) and hygromycin (in order to eliminate parental LTC-4D cells) . Twenty hybrid colonies were isolated individually using cloning rings and mass culture of hybrid cell colonies established using appropriate culture media and conditions, where necessary carrying out further purification by sub-cloning.

Cytogenetic analysis using high quality G-banding techniques was used to identify the X, Y, and T (4; X) marker chromosomes of the hybrids. These analyses showed that most of the hybrid cell lines carried nearly 4N chromosomes from the LTC-4D parental cell lineages and nearly 2N chromosomes from the T(4;X)37H parent (4N+2N, near-hexaploid hybrids) . Twelve clonally isolated hybrid cell lines were selected for further analysis; testing for LTC-4D-specific cell surface markers by flow cytometry and performing in vitro assays to determine multi-lineage differentiation potentials.

(b) The fusion of LTC-4D cells with cells derived from GMO5267 cell lines resulted many hybrid lines and we selected five clonally derived lines for cytogenetic analysis. The results of these analyses are presented in Table 4.

Table 4. Cytogenetic characterization of hybrid lines generated by fusing mouse mesenchymal stem cell line LTC-4D with mouse fibroblast GMO5267-derived cells

Note: The LTC-4D cell line contained two XX and two YY chromosomes and the GMO5267-derived cell line contained one X and one small marker chromosomes but no Y chromosome

Taken together, these results demonstrate that the intra- specific mouse hybrid cells containing near-hexaploid chromosome complements can proliferate in vitro without exhibiting the phenotype of cellular senescence. It should be noted that the original LTC-4D cell line contained near-tetraploid chromosome complements and this is probably why near-hexaploid hybrids were formed when they were fused with diploid or near-diploid fibroblasts. Our results demonstrate that by fusing 4N cells with 2N cells, near-hexaploid intra-specific hybrids can be generated. In this respect, our intra-specific hybrids differ from the intra- specific mouse hybrids generated by other workers who found near diploid chromosomes in the hybrids (Matveeva N.M. et al, 1998 "In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes". MoI Reprod Dev 50:128-38; Serov 0., et al, 2001 "Embryonic hybrid cells: a powerful tool for studying pluripotency and reprogramming of the differentiated cell chromosomes" An Acad Bras Cienc 73:561-8; Medvinsky A. & Smith A., 2003 "Stem cells: Fusion brings down barriers". Nature 422:823-5).

We have also generated three other series intra-specific mouse hybrids by fusing the non-transformed HygR T(4;X) fibroblasts with normal fibroblasts derived from three strains of knockout mice: (a) fusion of normal fibroblasts derived from p53-/- knockout mice with T(4;X)37H resulted in null-p53-T37 hybrids; (b) fusion of normal fibroblasts derived from Rb-/- knockout mice resulted in null-Rb-T37 hybrids, and (c) fusion of fibroblasts derived from heterozygote Rb+/- knockout mice resulted in Het-Rb-T37 hybrids.

All the hybrids were selected in medium containing G418 and hygromycin. These hybrid cells showed diverse morphologies as well as diverse chromosome (genomic) composition. The chromosome numbers of these hybrids varied from near 2N to near 6N. However, cells having flattened morphology and large size could be isolated by low density plating. Cells derived from the colonies with flattened morphology and large sizes contained chromosome numbers ranging from near tetraploid to near-hexaploid levels. It should be noted that while the fusion of mouse stem cells with mouse somatic cells results in hybrids containing near-diploid chromosomes (Matveeva N. M., et al, 1998 "In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes" . MoI Reprod Dev 50:128-38; Que J., et al, 2004 "Generation of hybrid cell lines with endothelial potential from spontaneous fusion of adult bone marrow cells with embryonic fibroblast feeder" In Vitro Cell Dev Biol Anim 40:143-149), fusion of normal mouse fibroblasts with normal fibroblasts results in near-tetraploid to near-hexaploid hybrids. It is possible that the chromosome composition of intra-specific mouse hybrids may vary depending on the tissue types used for generating cell hybrids. It should be emphasized however that the intra- specific hybrid cell lines comprising near-tetra and/or near- hexaploids can be generated by fusing a near tetraploid parental cell (for example the LTC-4D cell line) with a diploid parental cell. Example 8. Intra-specific human cell hybrids and inter-specific human-mouse cell hybrids

In a similar manner to the procedures described above, human fibroblasts cells were fused with a human osteoblast cell line in order to generate intra-specific human cell hybrids. The human osteoblast cell line CRL-11372 (earring the NeoR gene) was purchased from ATCC, USA. Before cell fusion, the hygromycin resistance gene was introduced into the fibroblast cells as described in Example 2. After fusion, hybrids were selected in culture medium containing G418 (in order to eliminate the fibroblast cells) and hygromycin (in order to eliminate the CRL-11372 cells) . Hybrid lines were characterized by cytogenetic analysis, which shows that they carry near-tetraploid to near-hexaploid chromosome complements. A selected set of hybrids were tested for genome reprogramming using molecular methods and assayed for multilineage differentiation, using a number of techniques known in the art.

By fusing cells derived from sub-populations of the mouse GMO5267 cell line with normal human fibroblasts, we have also generated inter-specific human-mouse cell hybrids containing near- tetraploid to near-hexaploid chromosome complements .

Claims

1. Method for the use of mammalian somatic cells, including cell hybrids, characterized by chromosomal aneuploidy (hyperploidy and/or hypoploidy) , for the purpose of extending the viability of differentiated cells by increasing numbers and rates of cell divisions and alleviating cessation of division of resting, damaged or senescent cells, including the controlled reprogramming of cell fate if/when required; comprising the generation by induced and/or spontaneous cell fusion and subsequent selection of cell hybrids containing heteroploid chromosome complements, including, but not limited to cells of hyper-tetraploid and hypo-and/or hyper-hexaploid chromosome complements .

2. Use, in accordance with claim 1, of cells including hybrids resulting from culturing or fusion of cells from the same or different cell lineages, derived from the same species of mammal; intra-species cell hybrids.

3. Use, in accordance with claim 1, of cells including hybrids resulting from culturing or fusion of cells from the same or different cell lineages of more than one species of mammal; inter-species cell hybrids.

4. Use, in accordance with any of the preceding claims, of resultant cells for the manufacture of a composition for transplantation to, or further fusion with or stimulation of, damaged tissue in order to facilitate tissue/organ regeneration .

5. Use, according to any of claims 1 to 3, of cell/hybrid cell culture, culture media, or individual factors purified there from, for the initiation and/or maintenance of cell proliferation in vitro.

6. Use, according to any of claims 1 to 3, of cell/hybrid cell culture, culture media, or individual factors purified there from, for the manufacture of a composition for the initiation and/or maintenance of cell proliferation in vivo, comprising bringing into contact said cells and/or media and/or purified growth promoting factors with damaged tissue/organs in order to facilitate tissue/organ regeneration.

7. Use, according to any of claims 1 to 3, of cells/hybrid cell culture, culture media, or individual factors purified there from, for the stimulation of cell proliferation and induction of cell differentiation in vitro.

8. Use, according to any of claim 1 to 3, of cell/hybrid cell culture, culture media, or individual factors purified there from, for the manufacture of a composition for the stimulation and/or regulation of cell differentiation in vivo, comprising bringing into contact said cells and/or media and/or purified differentiation factors with damaged tissue/organs in order to facilitate tissue/organ regeneration.

9. Use, for research, diagnostic and/or therapeutic purposes, of cells/hybrid cells generated by culture and/or cell fusion comprising autologous cell-types (i.e., both fusing parental cells derived from the same individual) , in accordance with claims 1 to 2 and claims 4 to 8.

10. Use, for research, diagnostic and/or therapeutic purposes, of cell culture and/or hybrid cell manipulations characterised by cell culture and cell fusions, comprising cells identical with or substantially similar to the hypo-diploid karyotype of cells derived from the GMO5267 cell line, in conjunction with any other cell type, derived from the same or different species, in accordance with claims 1 to 8.