Abstract:
The present invention provides a method of isolating a lineage specific stem cell in vitro, comprising a) transfecting a pluripotent embryonic stem cell with a construct comprising a regulatory region of a lineage specific gene operably linked to a DNA encoding a reporter protein, b) culturing the pluripotent embryonic stem cell under conditions such that the pluripotent embryonic stem cell differentiates into a lineage specific stem cell and c) separating the cells which express the reporter protein from the other cells in the culture, the cell which expresses the reporter protein being an isolated lineage specific stem cell. A lineage specific stem cell can also be identified utilizing this method.

Description:
BACKGROUND OF THE INVENTION 
     Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. 
     1. Field of the Invention 
     This invention relates to pluripotent stem cells and methods for isolating more committed progenitor cells from the pluripotent stem cells. 
     2. Background Art 
     Stem cells are undifferentiated, or immature cells that are capable of giving rise to multiple, specialized cell types and ultimately to terminally differentiated cells. These terminally differentiated cells comprise the fully functional organs and tissues within the adult animal and are the end product of embryonic development. Stem cells have two main characteristics. First, unlike any other cells, they are capable of dividing and differentiating into many different mature cell types within the body. Second, they are also able to renew themselves so that an essentially endless supply of mature cell types can be generated when needed. Because of this capacity for self-renewal, stem cells are therapeutically useful for the regeneration and repair of tissues. In contrast, terminally differentiated cells are not capable of self-renewal and are thus not capable of supporting regeneration and repair of damaged or diseased tissue. 
     The potency of a stem cell is measured by the number of different cell types it can ultimately produce. The most potent stem cell is the pluripotent stem cell (PSC) which can give rise to all cell types of the body (Wagner, E.; Matsui et at.; Resnick et al.). Other stem cells exist and include multipotent stem cells which give rise to two or more different cell types. For example, the multipotent hematopoietic stem cell is capable of giving rise to all cell types of the blood system (Jones et al.; Fleming et al.). Other known multipotent stem cells include a neuronal stem cell and a neural crest stem cell (Reynolds and Weiss; Stemple and Anderson). Bipotential stem cells are also considered multipotent stem cells since they give rise to more than one cell type. Specific examples of bipotential stem cells include the O-2A progenitor (Lillien and Raff; McKay, R.; Wolswijk and Noble) and the sympathoadrenal stem cell (Patterson, P. H.). There is one example of a monopotent stem cell, the epidermal stem cell (Jones and Watt). 
     The usefulness of stem cells for tissue regeneration and repair has been shown in several systems. For example, grafting of the hematopoietic stem cell has been shown to rescue an animal which has had its bone marrow subjected to lethal doses of radiation (Jones et al., supra). The O-2A progenitor has also been shown to remyelinate spinal cord neurons that have been chemically demyelinated (Groves et at.). 
     However beneficial these specific stem cells are, they still exhibit several practical drawbacks which limit their commercial development for biomedical applications. One disadvantage is their limited potency for developing into a broad range of cell lineages and tissues. Only the hematopoietic stem cell is capable of producing most cells within a tissue lineage, the other exhibit a very narrow range of developmental potential. Another disadvantage is the origin of the source material. Most neuronal stem cells have been isolated from newborn or early stage fetal tissue. The limited potency of these stem cells requires the independent isolation and maintenance of each cell type which is to be used for a specific application. Thus, the isolation, characterization and commercial usefulness of stem cells with other potentials will depend on the availability of large amounts of source material. 
     With the availability of a pluripotent stem cell, these disadvantages can be overcome if the pluripotent cell can be differentiated into more committed stem and progenitor cells. Differentiation into a stem cell with a desired potency and lineage specificity would allow an unlimited supply of source material and would also allow the treatment of a broad range of diseases due to the pluripotent nature of the stem cell. Such directed differentiation into a desired cell lineage would be very efficient and extremely cost effective for the commercial development of cellular therapeutics. However, because there are numerous differentiation pathways and points of commitment, and because the inductive effects are very complicated in the developing embryo, such directed differentiation of the pluripotent stem cell has not been accomplished in vitro. 
     To overcome the above limitations, those skilled in the art have resorted to time consuming experimentation or indirect methodologies in order to understand stem cell differentiation pathways and to isolate, through a series of in vitro and in vivo manipulations, more committed progenitor cells. For example, one of the most characterized stem cells is the bipotential 0-2A progenitor. It has been known for many years that this stem cell is capable of differentiating in vitro into either oligodendrocytes or type-2 astrocytes. However, it was only in recent years that the combination of growth factors needed to direct the differentiation down either pathway was fully understood. The sympathoadrenal stem cell is another such example where time consuming experimentation was necessary in order to understand its differentiation pathway. Although the differentiation pathways of these two bipotential stem cells are the most well characterized, the fact that it still took many years to understand their pathway exemplifies the problem of directed differentiation in vitro to obtain more committed progenitor or terminally differentiated cells. 
     There are also examples of the in vitro differentiation of multipotent and pluripotent stem cells. ES cells derived from blastocyst and post-implantation embryos can be allowed to uncontrollably differentiate into aggregates and embryoid bodies of terminally differentiated cells. Terminally differentiated cells within the aggregates and embryoid bodies comprise various cell types including extraembryonic endoderm, spontaneously contracting muscle, nerve and endothelial and fibroblast-like cells. ES cells can also be allowed to differentiate into cultures containing either neurons or skeletal muscle (Dinsmore et al.), or hematopoietic progenitors (Keller et al.; Biesecker and Emerson; Snodgrass et al.; Schmitt et al.). However, in none of these examples is the differentiation of the pluripotent stem cell directed down a particular pathway. Instead, they are allowed to differentiate randomly into a mixed population of terminally differentiated cells. Thus, there is no means of isolating a substantially pure population of progenitor cells of a desired cell lineage. 
     In order to obtain specific cell lineages differentiated from the pluripotent stem cell, those skilled in the art have relied on in vivo mechanisms to direct the differentiation into specific cell lineages. For example, (Otl et at., 1994), have described a method for isolating stem cells of the neuronal lineage after modifying pluripotent stem cells with a reporter construct and then reintroducing them into an early stage embryo. The reporter construct is expressed during neurogenesis and cells expressing the reporter gene are dissected out and placed in culture. Through in vivo mechanisms, this method allows for the isolation of cells committed to the neuronal lineage but, again, the dissected cells once placed in culture proceed to terminal differentiation. 
     Thus, there exists a need for a rapid method to differentiate and isolate more committed progenitor cells directly from stem cell cultures in vitro without undue experimentation. The present invention satisfies this need and provides related advantages as well. 
     SUMMARY OF THE INVENTION 
     The present invention provides a method of isolating a lineage specific stem cell in vitro, comprising: 
     a. transfecting a pluripotent embryonic stem cell with a construct comprising a regulatory region of a lineage specific gene operably linked to a DNA encoding a reporter protein; 
     b. culturing the pluripotent embryonic stem cell under conditions such that the pluripotent embryonic stem cell differentiates into a lineage specific stem cell; and 
     c. separating the cells which express the reporter protein from the other cells in the culture, 
     the cell which expresses the reporter protein being an isolated lineage specific stem cell. A lineage specific stem cell can also be identified utilizing this method. 
     DETAILED DESCRIPTION OF THE INVENTION 
     As used herein, &#34;lineage specific stem cell&#34; means a stem cell which is less potent than the pluripotent embryonic stem cell. In other words, the lineage specific stem cell has become developmentally committed to be a particular type of stem cell. For example, the pluripotent embryonic stem cell can be induced to be a neuronal stem cell. The neuronal stem cell can produce only neuronal cells. Thus, the cell is committed to the neuronal lineage and is a &#34;lineage specific stem cell.&#34; 
     As used here, &#34;pluripotent embryonic stem cell&#34; means a cell which can differentiate into any normal cell type including germ cells. Pluripotent embryonic stem cells are also referred to as embryonic germ cells depending on the method of isolation. 
     As used herein, &#34;transfecting&#34; means any manner of introducing a DNA construct into a cell for expression of the construct. Thus, transfection includes electroporation, lipofection, calcium phosphate mediated, DEAE dextran and the like. 
     &#34;Construct&#34; as used herein means any vector, such as a plasmid, phage or cosmid to which the DNA encoding the reporter protein and the regulatory region of a lineage specific gene have been inserted. The regulatory region is &#34;operably linked&#34; to the DNA encoding the reporter gene. Operably linked means the sequences are attached such that the reporter protein can be expressed in the cell and used as a means to isolate the cell. Various constructs can be created which are suitable for lineage specific stem cell isolation (Sambrook et at. and Ansubel et al.). 
     The &#34;regulatory region&#34; is the DNA which regulates the binding of transcription factors, including tissue specific and developmental state specific transcription factors, and controls the initiation of transcription. The size of the region depends on the lineage specific gene selected. 
     The methods encompassed by the technology entail tagging the pluripotent stem cells prior to differentiation with a reporter gene under the control of developmental and lineage specific enhancers. The type of reporter gene employed will depend on the desired goal of the experiment. For example, if it is necessary to follow the differentiation pathway of a specific lineage, or, to test the developmental specificity of the enhancer, then a reporter construct which allows tracking by visual observation is used in conjunction with a lineage specific enhancer (i.e., histochemistry). Typically, this experimental design is used for tracking and characterization of cell lineages and differentiation branch points. However, once lineages are characterized, this same system can be used for the isolation of lineage and stage specific stem cells by simply substituting the type of reporter gene from a histochemical marker to a surface membrane protein. Enhancer specificity will direct expression of the surface protein at the desired stage of isolation and fluorescent activated cell sorting (FACS) will allow the efficient isolation of the desired stem cell. Other immunological separation techniques such as panning may also be applicable for stem cell isolation. 
     A significant number of genes and their enhancers are known which direct the developmental and lineage specific expression of endogenous genes. The major candidate genes for early neuronal events such as floor plate induction are known. Lineage specificity of other genes is described further below. Therefore, the enhancer that will be used for expression will depend on what stem cell lineage and what stage of development is desired. In addition, as more detail is understood on the finer mechanistic distinctions of lineage specific expression and stem cell differentiation, it can be incorporated into the experimental protocol to fully optimize the system for the efficient isolation of a broad range of desired stem cells. 
     This technology allows the discovery and isolation of more restricted stem cells and committed progenitors for essentially any lineage in which there are characterized markers. There are several ways to use the molecular tagging method to isolate the lineage specific stem cell, or a committed progenitor within the same lineage and near the same stage of development. One alternative is to isolate the underrepresented population of lineage specific stem cells from a larger population of many cell types. The other alternative is to direct the differentiation of the pluripotent stem cell down the appropriate pathway (e.g., neuronal) to first enrich for a population of lineage specific stem cells. This latter alternative also ensures that short-range signalling which may be necessary for lineage specific stem cell formation is retained within the population. The advantage of the first approach is that it has the capabilities to rapidly obtain the desired cell. 
     Conditions which can be used to differentiate the pluripotent embryonic stem cell into various lineage specific stem cells is known in the art. For example, pluripotent embryonic stem cells can be allowed to aggregate in culture. Once the cells aggregate the natural communications between the cells causes certain of the cells to begin to differentiate down a particular lineage. The lineage specific cells can be separated at this point or, depending on the desired lineage, the aggregates can be allowed to form embryoid bodies. The lineage specific stem cells can then be isolated from the aggregates or embryoid body depending on the desired developmental stage of the stem cell. 
     Alternatively, the conditions used to differentiate the pluripotent embryonic stem cell can be performed by the addition of growth factors to the pluripotent embryonic stem cells in culture. These factors cause the cells to differentiate down a particular lineage depending on the growth factor added. For example, the factors which cause a pluripotent embryonic stem cell to differentiate into a hematopoietic stem cell are known to be contained in plasma derived serum (Keller et at.). The serum or isolated factors from the serum can, for example, be utilized to direct the pluripotent embryonic stem cell down the hematopoietic lineage. 
     Many methods are known in the art to separate a cell type based on the expression of a marker protein (Sambrook et al.). In a presently preferred embodiment, Fluorescent Activated Cell Sorting (FACS) is utilized (Fleming et al.). Various reporter proteins can be utilized including, for example, LacZ and proteins expressed on the cell surface. Depending on the marker utilized, various detecting methods can be utilized, e.g., immunoaffinity procedures, fluorescence, enzymes and the like. 
     Pluripotent embryonic stem cells can be obtained from established mouse cell lines (Hooper et at., 1987) or by following the methods set forth in Matsui et al. (1992). The method of Matsui et al. allows for the establishment of human pluripotent embryonic stem cells by utilizing the same methods substituting human growth and human fetal material of between six and twelve weeks. 
    
    
     The following example sets forth a specific embodiment of the invention. It should be recognized that other lineage specific regulatory regions from genes such as Dlx (Porteus et al.), Nlx (Price et al.), Emx (Simeone et al. EMBO J. 1992), Wnt (Roelink and Nuse), En (McMahon et al.), Hox (Chisaka and Capecchi; Lufkin et al.), acetylcholine receptor β chain (ACHRβ) (Otl et al.) and the like can be substituted for Otx (Simeone et al. Nature 1992; Otl et al.). Likewise, various reporter proteins, culture conditions and isolation methods can be substituted without departing from the scope of the invention. 
     EXAMPLE I 
     Construction of Stable PSC Lines and Isolation of Lineage Specific Neuronal Stem Cells 
     This Example shows the construction of PSC lines and isolation of neuronal specific stem cells that express a reporter gene under the control of a developmentally stage specific regulatory region of an early neuronal marker. 
     A. Construction of Stable Lines from Blastocyst Derived ES Cells 
     ES cell lines are constructed to express a reporter construct under the control of the Otx2 regulatory region. Otx2 is an early marker of neurogenesis. Differentiation into the neuronal lineage therefore results in the activation of the Otx2 regulatory region and biosynthesis of the reporter protein. The reporter protein used for isolation of early neuronal stem cells is the β-chain of the interleukin-2 (IL-2) receptor. This receptor was chosen because it is naturally expressed on the cell surface as a transmembrane protein and because specific antibodies are commercially available to the receptor. Expression of the IL-2 receptor on the surface allows isolation of the neuronal stem cells only after they have committed to the neuronal differentiation pathway. 
     The ES cell line used to construct the stable cell line is E14TG2a which was originally established by Hooper et al. (1987). ES cells are routinely cultured in DME supplemented with 15% FCS, 0.1 mM β-mercaptoethanol and 1,000 U hLIF on gelatin-coated plates. LIF is purchased from R&amp;D Systems (Minneapolis, Minn.). 
     To construct the reporter construct, which is also a targeting vector for homologous recombination, the vector contains two arms of homology to the IL-3 receptor gene. The 5&#39; arm corresponds to the first coding exon of the IL-2 receptor and the 3&#39; arm corresponds to the most 5&#39; sequences of the 5&#39; untranslated region sequence and regulatory region. Inserted upstream from the 5&#39; arm and in frame with the ATG initiation codon is the Otx2 regulatory region. Also, included as part of the insert is the neomycin resistance gene (neo R ) which is obtained from pMClneopolA (Stratagene, La Jolla, Calif.). The modified Herpes simplex virus thymidine kinase gene, HSV-tk, (Mansour et al.) is added to the 3&#39; end of the IL-2 receptor sequences, followed by the Bluescript vector (Stratagene). The final construct is termed OtxIL2R. 
     OtxIL2R is introduced into E14TG2a cells by electroporation. Briefly, ES cells are added to about 40 μg of Not I linearized targeting vector in 0.7 ml of culture medium using a BTX Transfector 100 at 250 V for 5 ms. Cells are plated at a density of 10 7  cells/90 mm petri dish and at 12 hr post electroporation. One plate is trypsinized and the number of cells counted. This number is used to calculate the cell survival. G418 is added to the remaining cultures at a concentration of 150 mg/ml as well as 2 μM gancyclovir (GanC) at 24 hr post electroporation to enable the positive-negative selection of the recombination event. One plate is selected in the absence of GanC to evaluate the electroporation and the enrichment factor of the GanC selection. The number of colonies without GanC selection is deduced relative to this control. 
     After 10 days in selection media, single colonies are picked and grown in duplicate. To screen for homologous recombination events, crude cell lysates from one of the duplicates are subjected to PCR analysis. PCR analysis is performed using 25-30 mer oligonucleotides which are complementary to sequences located in the neo R  gene and in the IL-2 receptor gene 3&#39; to the targeting vector sequences. PCR is performed using 100 ng/reaction of each primer, 2U Taq polymerase (Cetus Corp., Emeryville, Calif.) and crude cell lysate (about 10,000 cells/reaction; (Kim and Smithies). The thermocycling is performed as follows: 40 cycles consisting of denaturation at 93° C. for 1 min, annealing at a temperature between 42° and 59° C. for 30 sec and polymerization at 72° C. for 2 min. Screening is performed using pools of eight clones. The single clones contained in the pools with the expected PCR product are rescreened by PCR to identify the actual recombinants. Positive clones are expanded further for Southern blot analysis. Blot analysis is performed using 5 μg of digested DNA per lane loaded onto a 0.8% agarose gel and transferred to nitrocellulose. The filter is hybridized to a probe complementary to the coding sequence of the IL-2 receptor and analyzed for the appropriate size fragment (Sambrook et al. and Ansubel et al.). The OtxIL2R ES cells are then used for in vitro differentiation and isolation of lineage specific neuronal stem cells (see section C below). 
     B. Construction of Stable Lines from Primordial Germ Cells 
     Derived ES Cells 
     As with section A above, ES cell lines are constructed to express a reporter construct under the control of the Otx2 regulatory region. However, the ES cells used for these stable lines are derived from primordial germ cells of post implantation embryos (Matsui et al., supra). 
     Briefly, Sl 4  -m220 feeder cells are maintained in Dulbecco&#39;s modified Eagles&#39; medium (DMEM) with 10% calf serum and 50 μg/ml gentamycin. Cells are irradiated (500 rads) and plated at a density of 2×10 5  per well of a 24-well plate in the same medium 24 hrs before use. Wells are pretreated with 1% gelatin. To obtain primary cultures of primordial germ cells, ICR females are mated with (C57BL/6×DBA) males embryos and the caudal region of 8.5-12.5 days post coitum (dpc) embryos is dissociated into single cells by incubation at 37° C. with 0.05% trypsin, 0.02% EDTA in Ca 2+  /Mg 2  + -free Dulbecco&#39;s phosphate-buffered saline for about 10 min with gentle pipetting. Cells from the equivalent of 0.5 embryos are seeded into a well containing feeder cells and 1 ml of DMEM, 2 mM glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin and 100 μg/ml streptomycin and 15% FCS. Growth factors are added at the time of seeding, usually at the following concentrations, recombinant human LIF and bFGF (10-20 ng/ml) and soluble rat SF (60 ng/ml) and the medium is changed every day. Primary cultures are trypsinized and reseeded into wells containing Sl 4  -m220 feeder layers in PGC medium (above). For further subculture, rounded colonies of densely packed ES cells were carefully picked in a finely drawn pipette and trypsinized in a microdrop under mineral oil before seeding into wells containing feeder cells above. After several rounds of subculture the cells can be passaged without picking individual colonies. 
     The reporter construct used to stably transfect these ES cells is similar to that used with the blastocysts derived ES cells in that the reporter gene is a cell surface marker under the control of the Otx2 regulatory region. However, homologous recombination is not used to target the reporter gene to a specific locus and thus, only the neo R  gene is required as a selectable marker. The reporter gene used for early neuronal expression is also the β-chain of the IL-2 receptor, however it is truncated just after the membrane spanning sequence and fused in frame to an immunoaffinity tag (Affimax). Although the IL-2 receptor is inactive without the γ-chain, this design ensures the loss of normal function of the IL-2 receptor when used as a surface tag. 
     To construct the expression construct, the IL-2 receptor cDNA is truncated at a convenient restriction site and fused in frame with the immunoaffinity tag. Alternatively, the immunoaffinity tag can be incorporated at the desired location using site directed mutagenesis or by PCR mutagenesis. The IL-2 receptor/immunoaffinity tag sequences are then ligated into the appropriate reading frame of the pcDNAINeo expression vector (Invitrogen, San Diego, Calif.). The promoter sequences of the expression vector are substituted with the Otx2 regulatory region for state specific expression of the reporter protein. The final construct is termed OtxIL2Af. Transfection into the above ES cells is performed by calcium-phosphate mediated transfection. Neomycin resistant colonies are picked, expanded and screened by PCR and RNase protection assay for intact OtxIL2Af construct sequences. All procedures described are well known to those skilled in the art and can be found in common laboratory manuals such as Sambrook et al. and Ansubel et at., supra. The OtxIL2Af ES cell lines are then used for in vitro differentiation and isolation of lineage specific neuronal stem cells (see section C below). 
     C. Isolation of Lineage Specific Neuronal Stem Cells Either the ES OtxIL2R or the ES OtxIL2Af cell lines are used in the methods below to isolate early stem cell of the neuronal cell lineage. Briefly, for in vitro differentiation into aggregates or embryoid body formation, ES cells are plated at a density of 10 7  cells/100 mm bacterial petri dish in DMEM supplemented with 10% FCS and 0.1 mM β-mercaptoethanol. Culture medium is changed every day. 
     Isolation of early neuronal stem cells is performed by FACS isolation of the differentiating cultures taken at various time points. By 5 to 7 days in culture most of the aggregates differentiate into typical simple or cystic embryoid bodies with a clear outer layer of extraembryonic endoderm cells. When the embryoid bodies are returned to tissue culture plastic dishes they rapidly attach and give rise to a variety of cell types including extraembryonic endoderm, spontaneously contracting muscle, nerve and endothelial and fibroblast-like cells. Time points are taken both before (0-5 days) and after (6 14 days) returning to tissue culture plastic. 
     FACS isolation was performed using methods well known in the art. Briefly, cell aggregates are dissociated into single cells by treatment in Ca 2+  /Mg 2+  -free Dulbecco&#39;s phosphate-buffered saline plus 54 mM EDTA and washed in DMEM containing 10% FCS. The cells are then incubated on ice with an equal volume of a 1:50 dilution of the appropriate antibody (1×10 6  cells in 0.1 ml) for 30 rain followed by a 10 ml wash in ice cold DMEM containing 10% FCS. Fluorescent conjugated secondary antibody is added at a dilution of 1:100 (0.1 ml) and again incubated on ice for an additional 30 min. The cells are washed once with 10 mls of ice cold DMEM containing 10% FCS and subjected to FACS isolation. Collected cells are plated in media allowed to proliferate. To prevent progression into terminally differentiated cell types various growth factors are assessed to maintain the potency of the early neuronal stem cells. 
     Although the invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. 
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