United States Patent: 6,780,641
Issued: August 24, 2004
Inventors: Kim; Seung U. (Vancouver, CA)
Assignee: University of British Columbia (Vancover, CA)
Appl. No.: 887145
Filed: June 22, 2001
An immortalized human cell line is provided which has the characteristics of human embryonic microglia. Such immortalized microglia cells express CD68, CD11c and MHC class I and II antigens as surface markers; have demonstrable phagocytic properties; and produce progeny continuously while maintained in culture. A method of transforming human microglial cells into an immortalized cell line is also provided. The genetically modified human microglia cells can express active substances from a selected group consisting of MIP-1.beta., MCP-1, IL-1.beta., IL-6, IL-12, and IL-15; and in the stimulated state can overexpress at lest cytokines, chemokines, and other cytotoxic and neurotoxic substances. Such immortalized microglia cells can be used for screening of compounds for diseases. These cells may be utilized for the treatment of at least Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis, stroke, spinal cord injuries, ataxia, autoimmune diseases and AIDS-dementia.
SUMMARY OF THE INVENTION
The present invention has multiple aspects. A primary aspect of this invention provides a genetically modified human microglia cell maintained as a stable cell line in-vitro comprising:
a modified microglia cell of human origin which
(i) has demonstrable phagocytic properties;
(ii) produces progeny continuously while maintained in culture;
(iii) presents at least CD11b and CD68 as surface antigens; and
(iv) contains human genomic DNA which has been genetically modified to include a viral vector carrying at least one DNA segment encoding an exogenous gene for intracellular expression.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is the establishment and characterization of several continuous cell lines of immortalized human microglia, labeled as HMO6, generated by transfection of embryonic (fetal) human microglia (HM) with a retroviral vector containing cDNA for the v-myc oncogene. The invention provides a phenotypic characterization of these immortalized human microglia; and discloses the expression of cytokines and chemokines following exposure to .beta. amyloid peptides using HM and HMO6 cells. For a clearer understanding and better appreciation of the subject matter as a whole which comprises the present invention, the detailed description will be presented as separate sections.
I. A Preferred Method for Producing Immortalized Human Microglia Cells and Continuous Cell Lines
`Human microglial cell line, as used herein, means a human-derived cell line with microglial characteristics, including at least the specific antigens CD68 and CD11b. Also, as used herein, "non-fetal" refers to the fact that the progeny cells are expanded from immortalized cells in-vitro, and there is no need to return to an embryonic/fetal source for additional microglia cells.
It should be understood that a number of different vectors besides the retroviral vector described herein can be used to transform embryonic human microglia (HM) cells. The transformation of such embryonic HM cells is conventionally known and routinely performed within the art; and HM cells transformed by any other vector can be prepared as disclosed below and subsequently tested to assure the presence of identifying microglial phenotypic markers. Further, the vectors are not limited to immortalizing HM cells with the oncogene v-myc. Many other varieties of oncogenes are known in the art and are suitable for immortalizing embryonic human microglia.
Standard molecular biology techniques known in the art and not specifically described herein are generally followed as in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989). Polymerase chain reaction (PCR) is carried out generally as in PCR Protocols: Guide to Methods and Applications, Academic Press, San Diego, Calif. (1990). Reactions and manipulations involving other nucleic acid techniques, unless stated otherwise, are performed as generally described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory Press, and the methodology set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659; and 5,272,057 and incorporated herein by reference. In-situ PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences [Testoni et al., Blood 87: 3822 (1996)].
Standard methods in immunology known in the art and not specifically described herein are generally followed as in Stites et al., Basic and Clinical Immunology, 8th Ed., Appleton & Lange, Norwalk, Conn. (1994); and Mishell and Shigi (eds.), Selected Methods in Cellular Immunology, W. H. Freeman and Co., New York (1980).
Human Microglia Culture
Brain tissue (mostly telencephalon) is obtained from 12-18 weeks gestation embryos. The brain tissue is incubated in phosphate buffered saline (PBS) containing 0.25% trypsin and 40 ug/ml DNase for 30 min. at 37oC. and then dissociated into single cells by gentle pipetting. Dissociated single cells are then preferably grown in a feeding medium consisting of Dulbecco's modified Eagle's medium (DMEM) to which the following are added: 5% horse serum, 5 mg/ml D-glucose and 20 ug/ml gentamicin. After 2-3 weeks of growth in flasks, free-floating microglia can be collected and plated in 6-well plates coated with poly-L-lysine.
It is also desirable that, for the last part of this time, microglia are exposed to granulocyte macrophage colony stimulating factor (GM-CSF) at final concentration of 8 ug/ml for 9-12 days with a medium change every 3 days. GM-CSF treatment stimulated proliferation of microglia results in a 3-4 fold increase in microglial population.
The human microglia isolated from primary cultures of embryonic human telencephalon brain cells are mostly round cells, with filopodia surrounding the cell bodies, or appear as slender cells having several primary branches. The cells typically are from 8-12 um in size. Human microglia composed in excess of 98% of plated cells (as determined using the specific cell type-specific marker, ricinus communis agglutinin-1 and CD11b).
Retrovirus-Media Gene Transfer
Embryonic human microglia grown with GM-CSF for 9-12 days are then subjected to retrovirally mediated transduction of v-myc and subsequent cloning. An amphotropic replication-incompetent retroviral vector encoding v-myc oncogene transcribed from mouse leukemia virus LTR plus neomycin-resistant gene transcribed from an internal SV40 early promoter is preferred for use to infect human microglia and induce propagation of immortalized human microglia cell lines. This amphotropic vector, PASK 1.2, was generated using the ecotropic retroviral vector encoding v-myc [obtained from the American Type Culture Collection (ATCC), Rockville, Md.] to infect the PA317 amphotropic packaging line.
Successful infectants were then selected and expanded. Transfection of embryonic human microglia in 6-well plates is preferably performed by the following procedures: 2 ml of supernatant from the PA317 packaging line and 8 ug/ml polybrene is added to microglia in 6-well plates and incubated for 4 hr at 37oC.; the solution is then replaced with fresh feeding medium; and transfection repeated 24 hr later. After 72 hr, the transfected cells are selected with neomycin (G418). Individual clones are generated by limiting dilution and propagated further.
In this manner, several neomycin (G-418) resistant colonies were isolated, expanded and were named HMO6. One of these HMO6 cell lines, the HMO6.A1 cell line, has been investigated for its cellular and molecular characteristics, as presented hereinafter. The terms "HM06" and "HM06.A1" are therefore synonymous and are used interchangeably hereinafter.
II. Characteristics and Properties of Immortalized Human Microglia Cells
The HMO6 human microglia cell and cell line is presented as a typical representation and illustrative example of the immortalized cells constituting the present invention. For these reasons, the detailed description will focus upon and be limited to characterizing the attributes of the HMO6 cell.
An immunohistochemical study (presented in detail by the Experiments described hereinafter) utilized a range of antibodies to identify those surface antigens which are expressed by the immortalized microglia cells. A summary listing of such surface markers is given by Table 1 below.
TABLE 1 Cell Type-Specific Markers For CNS Cells Antigen Identifying Specificity HMO6 Human mitochondria mitochondria + Pan-myc myc + RCA-1 lectin microglia ++ CD11b microglia + CD68 microglia + HLA-ABC MHC class I + HLA-DR MHC class II + MAP-2 neuron - .beta. tubulin III neuron - GFAP astrocytes - MBP oligodendrocytes -
Antigen reactivity were determined by immunohistochemistry. Immunoreactivity was expressed in a scale ranging from - (negative) to ++ (strong positive).
As the data of Table 1 shows, HMO6 cells expressed surface antigens specific for microglia cells such as CD11b, CD68, HLA-ABC, HLA-DR, and ricinus commuriis agglutinin-1 lectin (RCA-1) as determined by immunocytochemistry (Antibody source: DAKO Diagnostics Canada, Inc., Mississauga, Ontario).
When HMO6 cells were exposed for two hours to medium containing latex beads (0.8 um size, Sigma) at the ratio of 1 .mu.l per 1 ml medium or to medium containing carbon particles (prepared from Chinese ink stick) at 37oC., all cells became loaded with latex beads or carbon particles. This demonstrates that the HMO6 cells are capable of active phagocytosis, one of the most significant function and features characterizing microglia-macrophage lineage cells of the CNS.
Chemokine and Cytokine Expression
To determine if and what pharmacologically active molecules are expressed and produced by HMO6 cells in a non-stimulatory condition (i.e., without addition of or reactive contact with active stimulating agents such as TNF-.alpha., LPS, and the like), a series of RT-PCR analyses were performed. Detailed description of such RT-PCR analyses themselves is provided by the Experiments hereinafter. The results of these analyses, however, are listed in summary form by Table 2 below.
TABLE 2 RT-PCR Analysis of Non-Stimulated HMO6 Cells Constitutive marker/expression of IL-1b + Constitutive marker/expression of IL-6 + Constitutive marker/expression of IL-8 + Constitutive marker/expression of IL-12 + Constitutive marker/expression of IL-15 + Constitutive marker/expression of TNF-.alpha. -* Constitutive marker/expression of MIP-1.alpha. -* Constitutive marker/expression of MIP-1.beta. + Constitutive marker/expression of MCP-1 + *Expression of TNF-.alpha. and MIP-1.alpha. was induced by treatment with TNF-.alpha. or A.beta..
In comparison, HMO6 cells can be stimulated using a variety of active agents. A summary of the expressed active molecules generated by specific stimulating agents is given by Table 3 below.
TABLE 3 RT-PCR Analysis Of Agent Stimulated HMO6 Cells Stimulated HMO6 cell Stimulating expressed active agent substance(s) TNF-.alpha. TNF-.alpha. MIP-1.alpha. IL-1.beta. None LPS IL-8 IL-12 MIP-1.beta. P2y2 A.beta.25-35 TNF-.alpha. Elevated IL-12 CXCR4 P2y2 IFN-.gamma. P2y2 B7-2
III. Expected and Intended in-vitro Assay Uses and in-vivo Therapeutic Uses for the Immortalized Human Microglia Cells
It has been long recognized that microglia play a critical role as resident immunocompetent cells and phagocytic cells in the central nervous system (CNS) and serve as scavenger cells in the event of infection, inflammation, trauma, ischemia and neurodegeneration in the CNS. Activated microglia have been observed in pathological lesions in neurodegenerative diseases and are believed to be involved in initiation or progression of CNS pathology. Neurodegeneration is often preceded or is concomitant with the functional responses of microglia in-vivo, including cell proliferation and the secretion of active molecules including oxygen radicals, proteases and pro-inflammatory cytokines.
Moreover, recent studies have indicated that the activation of native microglia precedes or is concomitant with neuronal and glial cell degeneration in a variety of neurological diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD), Parkinson disease, Huntington disease (HD), stroke, brain trauma, AIDS-dementia, and multiple sclerosis. Thus, microglia-mediated neurotoxicity appears to be critical in tissue damage and neuronal death during the initiation and pathological progression of neurological disorders, in-vivo.
In addition, recent experimental studies reported by others indicate that murine microglia cells produce neurotrophic factor(s) that promote survival of CNS neurons in an environment in which majority of neurons undergo degeneration in the absence of microglia conditioned medium. Previous studies have also indicated that murine/human microglia cells in culture produce neurotoxic substance following exposure to .beta.-amyloid for 24-48 hours.
For these reasons, a variety of in-vitro assays and in-vivo therapeutic uses are envisioned and intended for the present invention. These include the following:
1. The immortalized human microglia cells can be used in-vitro to isolate neurotoxic or neurotrophic molecules naturally produced by human microglia or produced in response to inflammatory factors or neuroactive molecules such as .beta.-amyloid.
2. Because microglia have been implicated in neurological disorders, such as Alzheimer disease, Parkinson disease, AIDS-dementia, ALS and MS, the immortalized human microglia can be used for discovery (screening) of new drugs to treat the aforementioned conditions and inflammation. Prospective drug candidates are those that can counter or reduce production of proinflammatory cytokines, oxygen radicals, proteases such as caspase-3 and -8, and neurotoxic agents such as .beta.-amyloid.
3. Human immortalized microglial cells can be further genetically manipulated to express and produce additional proteins, peptides, or prodrugs. Such substances would include a diverse range of chemokines, cytokines, and various marker proteins (e.g., LacZ and GFP), growth factors, neurotrophic molecules, anti-apoptotic molecules (e.g., Bcl-2), and enzyme inhibitors (e.g., caspase inhibitor). Microglia cells can be additionally genetically modified to block the production of proteins that typically become overproduced by nervous system pathologies. For example, upstream from the v-myc gene, there can be inserted an activatable suppressor gene. Alternately, for human treatment, there can be inserted a suicide gene.
4. Microglia cells are known to be activated by inflammatory factors/cytokines/chemokines in neurodegerative diseases including Alzheimer disease, Parkinson disease, Huntington disease and ALS, and stroke. In Alzheimer disease, nonsteroidal anti-inflammatory drug (NSAID) such as indomethacin has been reported to delay progression of disease process by blocking/inhibiting microglial activities. For that reason, human immortalized microglia cells could be utilized for screening drugs which can block or suppress microglial activation and progression of disease process.
5. It has been observed that murine microglia cells introduced via venous injection could translocate into brain parenchyma. It is well known that external molecules or cell elements can not pass barrier called blood-brain-barrier (BBB) and enter into brain proper. For that reason, microglia cells capable of bypassing BBB could deliver drugs into brain without difficulty. One can also genetically engineer immortalized human microglia cells to carry desired gene to produce proteins and/or prodrugs and then introduced into patients brain. This is a novel and useful way of drug delivery system.
In the expected therapeutic methods of the present invention, the immortalized human microglia can be administered in various ways as would be appropriate to implant in the central nervous system, including but not limited to parenteral administration, intrathecal administration, intraventricular administration, and intranigral administration.
In each envisioned and intended in-vivo context, and particularly with the therapeutic treatment of neurodegenerative diseases and disorders, the cells of the present invention are to be administered and implanted in accordance with accepted good medical practices. Such good medical practices take into account the clinical condition of the individual patient; the site and method of administration; scheduling of administration; and patient age, sex, body weight and other personal factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such medical considerations as are known in the art. The cell amount must be sufficient to achieve clinical improvement, including (but not limited to) improved survival rate or more rapid recovery; and improvement or elimination of clinical symptoms and other medical indicators as are recognized by those skilled in the medical arts.
Claim 1 of 10 Claims
What I claim is:
1. A genetically modified human microglia cell which can be maintained as a stable, substantially homogeneous cell line in-vitro, said genetically modified cell comprising:
a microglia cell of human origin which is stable when maintained in culture and which
(i) has demonstrable phagocytic properties;
(ii) produces substantially homogenous progeny continuously while maintained in culture;
(iii) presents at least CD11b and CD68 as surface antigens; and
(iv) contains human genomic DNA which has been genetically modified to include a viral vector carrying at least one DNA segment encoding an exogenous gene for intracellular expression wherein said viral vector is an amphotrophic retroviral viral vector and wherein said viral vector includes an exogenous DNA sequence encoding a v-myc gene.