(en)The present invention provides in vitro methods of expansion and propagation of undifferentiated progenitor cells and more specifically undifferentiated progenitor cells containing Islet1, a marker apparently unique to proliferating cardiac stem cells. Methods are described for isolation of stem cell populations as well as for provoking expansion and propagation of undifferentiated progenitor cells without differentiation, to provide cardiac repair or improve cardiac function, for example.

1.ApplicationNumber: US-54405304-A
1.PublishNumber: US-7745113-B2
2.Date Publish: 20100629
3.Inventor: EVANS SYLVIA M.
CHEN JU
CAI CHENLENG
MORETTI ALESSANDRA
CHIEN KENNETH R.
LAUGWITZ KARL-LUDWIG

4.Inventor Harmonized: EVANS SYLVIA M(US)
CHEN JU(US)
CAI CHENLENG(US)
MORETTI ALESSANDRA(US)
CHIEN KENNETH R(US)
LAUGWITZ KARL-LUDWIG(US)

5.Country: US
6.Claims:
(en)The present invention provides in vitro methods of expansion and propagation of undifferentiated progenitor cells and more specifically undifferentiated progenitor cells containing Islet1, a marker apparently unique to proliferating cardiac stem cells. Methods are described for isolation of stem cell populations as well as for provoking expansion and propagation of undifferentiated progenitor cells without differentiation, to provide cardiac repair or improve cardiac function, for example.

7.Description:
(en)CROSS REFERENCE TO RELATED APPLICATIONS
This application is a 35 USC §371 National Stage application of PCT Application No. PCT/US2004/002978 filed Feb. 2, 2004; which claims the benefit under 35 USC §119(e) to U.S. Application Ser. No. 60/444,247 filed Jan. 31, 2003, and U.S. Application Ser. No. 60/484,809 filed Jul. 2, 2003. The disclosure of each of the prior applications is considered part of and is incorporated by reference in the disclosure of this application.


GRANT INFORMATION
This invention was made with government support under Grant No. HL66276 awarded by the National Institutes of Health. The United States government has certain rights in this invention.


FIELD OF THE INVENTION
The invention relates generally to in vitro expansion and propagation of undifferentiated progenitor cells and more specifically to undifferentiated progenitor cells containing Islet1.
BACKGROUND INFORMATION
Congenital heart disease is the most common of all birth defects (Hoffman and Kaplan, 2002). For successful prevention of or therapeutic intervention in congenital heart disease, it is of utmost importance to understand its etiology. Toward this goal, an understanding of the origin of specific cardiac lineages and their interactions with each other is critical. Understanding the origin and properties of cardiac progenitors is also important for the development of cardiac stem cell therapies for both congenital and adult heart disease.
Recent work has defined two fields of cardiac progenitors, dubbed the primary and secondary, or anterior heart fields (Kelly and Buckingham, 2002). The primary heart field is believed to give rise to the atria and ventricles of the heart, while the secondary or anterior field is believed to give rise to the outflow tract. The secondary field is believed to reside anterior and dorsal to the heart at the early linear heart tube stage. Initial evidence that the outflow tract of the heart was not present in the linear heart tube came from a series of in vivo lineage studies performed in chick embryos by de la Cruz and colleagues from 1977 onward (de la Cruz, 2000). These studies demonstrated that the outflow tract was not present at the linear heart tube stage, but did not indicate where the outflow tract came from at a later stage.
Recently, the source of the outflow tract has been addressed by studies from three different laboratories, two performed in chick embryos, and one performed in mouse embryos (Kelly et al., 2001; Mjaatvedt et al., 2001; Waldo et al., 2001). Results of these studies demonstrated that some cells in the outflow tract originate from splanchnic mesoderm adjacent to the pharyngeal endoderm. The extent of the contribution, and the boundaries of the “secondary” or “anterior” heart field could not be definitively assessed from results of these experiments.
Stem cells have been defined in many different ways. However, the main principles include: (1) self-renewal, or the ability to generate daughter cells with characteristics similar to the initiating mother cell; (2) multi-lineage differentiation of a single cell; and (3) in vivo functional reconstitution of damaged tissue.
The Embryonic Stem (ES) cells, first obtained from mouse (Evans and Kaufmann, 1981) and more recently from non-human primates and human blastocysts (Thomson, et al., 1998), display all three characteristics. ES cells are pluripotent cells derived from the inner cell mass of the blastocyst that can be propagated indefinitely in an undifferentiated state. Both mouse and human ES cell-lines have been maintained continuously in culture for more than 300 cell doublings. ES cells differentiate into all somatic cell types when injected into a blastocyst and form mature progeny cells of all three embryonic germ layers in vitro. Finally, all differentiated progeny of ES cells are functional cells, as mice generated by tetraploid embryo complementation are viable. Although ES cells have been isolated from humans, their use in research as well as their therapeutic potential is encumbered by ethical considerations.
Most adult stem cells also fulfil the stem cell criteria mentioned above, even though the degree of self-renewal and differentiation is less than that seen for ES cells. The best studied adult stem cell, the hematopoietic stem cell (HSC) (Weissman, 2000), undergoes in vivo self-renewing cell divisions, differentiates at the single cell level into all mature blood elements, and functionally repopulates the bone marrow of myeloablated animals and humans. Other adult stem cells have been more recently defined and are, therefore, less well studied. However, neural stem cells (NSC) (Gage, 2000), mesenchymal stem cells (MSC) (Jiang, et al., 2002) and epidermal stem cells (Toma, et al., (2001) all fulfil these basic criteria. Other cells also termed stem cells, such as angioblasts or endothelial stem cells (Rafii, et al., 1994), display all the required characteristics, except that they differentiate only into a single type of cells.
Over the last few years a plethora of literature has been published indicating that cells from a given tissue might be capable of differentiating into cells of a different tissue “Stem cell plasticity” is a new term that has been used to describe the recent observations that greater potential might persist in postnatal adult stem cells than previously expected. The majority of studies using bone marrow (BM), or peripheral blood enriched for HSC were based on in vivo transplantation of either sex-mismatched cells or genetically marked cells, and detection of donor cells was based on the presence of the Y-chromosome or the marker gene. There are excellent reviews of the pitfalls involved in the detection of donor cells using either marking system (Tisdale and Dunbar, 2002). Differentiation, not only into hematopoietic cells, but also into cells with characteristics of skeletal muscle (Gussoni, et al., 1999), cardiac muscle (Orlic, et al., 2001), endothelium (Jackson, et al., 2001), neuroectoderm (Brazelton, et al., 2000) and endodermal cells (Krause, et al., 2001), including hepatocytes, has been described.
In 80% of these studies, fresh BM cells were transplanted without prior in vitro culture, so that the question of whether the cell with plasticity can undergo self-renewal could not be assessed. In the majority of these studies, non-purified populations of cells or cells purified to partial homogeneity were transplanted, therefore making it impossible to study the clonal origin of differentiated cells or the tissue of origin of cells with characteristics of a second tissue. Finally, these studies depended on phenotypic characteristics to define differentiation into cells different from the tissue of origin, but have yet to demonstrate that the cells of the second tissue have functional characteristics of that lineage.
Thus there is need in the art for new and better methods of in vitro expansion and propagation of undifferentiated cardiac progenitor cells. The method includes culturing isolated undifferentiated progenitor cells that express Islet1 under conditions sufficient for progenitor cell growth. study of
SUMMARY OF THE INVENTION
Analysis of mice lacking Islet1, a LIM homeodomain transcription factor, has revealed a new paradigm for heart development. Hearts of islet1 knockout mice are completely missing the outflow tract, right ventricle, and much of the atria. Islet1 expression and lineage tracing of islet1-expressing progenitors demonstrate that Islet1 is a marker for a distinct population of undifferentiated cardiac progenitors that give rise to cardiac segments found missing in isl 1 mutants. Islet1 function is required for these progenitors to contribute to the heart. In islet1 mutants, islet1 expressing progenitors are progressively reduced in number, and bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs) are down regulated. The studies described herein define two cardiogenic fields, of which one expresses and requires Islet and the other does not. The results of these studies have implications for the development of specific cardiac lineages, cardiac looping, left right asymmetry, cardiac evolution, and cardiac stem cells.
Accordingly, in one embodiment, the invention provides a method for detecting a stem cell comprising determining expression of Islet1 nucleic acid or expression product in a cell.
In another embodiment, the invention provides a method for isolating or enriching for stem cells comprising contacting the cells with an agent reactive with Islet1 and separating the reactive positive cells from reactive negative cells, thereby isolating or enriching for stem cells.
In yet another embodiment, the invention provides methods for generating a stem cell comprising contacting an undifferentiated progenitor cell that expresses Islet 1 with an agent that activates or enhances expression of Islet1 in the cell so as to activate or enhance expression of Islet 1 in the cell.
In still another embodiment, the invention provides an in vitro method for expansion and propagation of undifferentiated cardiac progenitor cells. The method includes culturing isolated undifferentiated progenitor cells that express Islet1 under conditions sufficient for progenitor cell growth. In the method, the conditions sufficient for progenitor cell growth include culturing the cells on a feeder layer of species-specific cardiac fibroblasts or conditioned medium of fibroblasts from the heart.
In another embodiment, the invention provides a composition that is an enriched population of Islet1 positive stem cells comprising greater than 90% Islet1 positive stem cells, as compared with other cell types.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is the mRNA sequence of an EST of Islet 1 (SEQ ID NO:1).
FIG. 2 is the DNA sequence of Islet 1 (mouse) available through GenBank under accession number: NM — 021459 XM — 354773. (SEQ ID NO:2).



DETAILED DESCRIPTION OF THE INVENTION
It has been discovered that Islet1 (SEQ ID NO: 2) is a transcription factor that is a unique marker for proliferating cardiac stem cells ( FIG. 2 ). It is the only gene known to date that is specifically expressed in cardiogenic stem cells, but not in differentiated cardiac cells. Islet1 may be a master regulator of the cardiogenic stem cell state. This discovery enables use of islet1 expression as a means to isolate endogenous cardiogenic stem cells, or to create cardiogenic stem cells. Islet1 is also expressed in other progenitor, or “stem cell” populations, including those of the pancreas, the neural crest, the aorta-gonad-mesonephros region (hematopoietic and endothelial progenitors), and other cell types. This expression, in concert with data described herein pertaining to cardiogenic stem cells, shows that islet marks, not only cardiogenic stem cells, but other pluripotent stem cells as well.
No other gene is known that is specifically expressed in undifferentiated cardiogenic precursors at earliest stages. Islet1 is a unique identifier of this cell population. Islet1 is also required for these precursors to contribute to development of the heart. In islet1 mutants, cardiogenic lineages normally derived from islet1-expressing progenitors are absent. Thus, Islet is unique in being expressed in a number of embryonically distinct pluripotential progenitors. Islet is a transcription factor that drives the stem cell state.
Utilizing islet1 as a marker, cells can be isolated from early embryos, hybridized with fluorescently labelled islet1 antibodies, and sorted for stem cells by FACS. Alternatively, genes (eg. lacZ, GFP, cre) can be inserted into the endogenous islet1 locus and used as a basis of cell identification or sorting. Cardiogenic stem cell lines can be created by expressing islet1 alone or in combination with Nkx2.5, another transcription factor that is expressed in cardiac progenitors, but is also expressed in differentiated cardiac cells. To differentiate these cardiogenic precursors, islet1 expression was downregulated by genetic means or by application of growth factors. Other stem cell lines can be created in a similar manner, expressing islet1 alone or in combination with other factors specific to distinct lineages, to create pluripotent cells, which can differentiate to multiple lineages, or specific lineages dependent on the genetic or physical environment.
A large body of data attests to the conclusion that islet1 is a marker of cardiogenic stem cells prior to their differentiation. This finding has led to the concept of utilizing islet1 as a means to isolate cardiogenic stem cells, from embryonic, neonatal or adult stages from a variety of experimental model animals, and from humans. An islet1 antibody can be obtained to detect these cell populations, and a mouse line in which GFP has been inserted into the islet1 locus, using known techniques, can be created. It has also been determined that islet is expressed in the aorta-gonad-mesonephros region, a region critical for generating multipotential hematopoietic and endothelial precursors.
Several therapeutic applications arise from the studies described herein utilizing these stem cells. For example, methods are provided for converting distinct cell types into cardiogenic stem cells for therapeutic application: e.g., isolation of skin fibroblasts or bone marrow stem cells from a cardiac patient, converting these cells to cardiogenic cells, and then injecting the converted cells into the patient. This method can be used to treat cardiac diseases, including post-infarct, heart failure, ischemic heart disease. Other applications include provoking proliferation and/or differentiation of resident cardiogenic stem cells within the differentiated heart to provide cardiac repair or improve cardiac function. Isolated cardiogenic stem cells can be used for therapeutic drug screening, for toxicology studies and for tissue engineering. In all of these procedures, other distinct cell lineages that may be derived from islet positive stem cells can also be used, with applications to relevant human disease.
The invention is based on the discovery that boundaries of the two cardiogenic fields are different than previously expected. One progenitor population expresses islet1 and will give rise to the outflow tract, right ventricle, a subset of left ventricular cells, and a majority of atrial cells. The other does not express islet1, and will give rise to a subset of the left ventricular cells and some atrial cells. The specific expression of islet1 in undifferentiated precursors also allows, for the first time, a precise visualization of the islet1 expressing progenitor population, and gives us an important handle for the isolation and characterization of a cardiac stem cell population. Islet1 not only defines this stem cell population, but is also required for these cells to contribute to the heart, providing the first genetic evidence for distinct heart fields.
Islet1 (Isl 1) knockout mice have been examined for defects in both motor neuron and pancreatic development (Ahlgren et al., 1997; Pfaff et al., 1996). Mice which are homozygous null for isl 1 exhibit growth retardation at approximately ED9.5, and die at approximately ED10.5. Heterozygous mutants survive, and have no apparent phenotype. The cause of death in homozygous mutants has not previously been addressed, although vascular abnormalities were suspected (Pfaff et al., 1996). Therefore the cause of death in isl 1 −/− mice was examined.
When homozygous null embryos between ED9.0 to ED9.5 were examined, severely abnormal hearts were found. At a gross morphological level, mutant hearts appeared to consist of a single, misshapen, and undivided chamber. Histological analysis confirmed this impression. As an initial attempt to characterize chamber identity of cells within mutant hearts, whole mount in situ hybridization analysis with markers for cardiac chambers was performed. Ventricular myosin light chain 2 (MLC2v) mRNA specifically marks ventricular cells, and cells of the A/V junction (Franco et al., 1999). At these stages, atrial myosin light chain 2 (MLC2a) mRNA marks all myocardial cells (Kubalak et al., 1994). Hybridization with probes for MLC2v and MLC2a mRNAs demonstrated that cells within the anterior part of the single chamber had ventricular identity, whereas cells in the posterior part of the single chamber did not, and were therefore likely to have atrial identity.
A number of transcription factors are regionally expressed within the heart, and a panel of these was used to further explore cellular identity within isl 1 mutant hearts. At stages examined, tbx5 is specifically expressed in the posterior pole of the heart, in atria and left ventricle (Bruneau et al., 1999). In islet1 mutants, both atrial and ventricular segments of the heart expressed tbx5, indicating that ventricular portions of the mutant heart had left ventricular identity. EHand is expressed in left ventricle, but not right ventricle (Cross et al., 1995; Cserjesi et al., 1995; Thomas et al., 1998). In isl 1 −/− embryos, EHand was expressed throughout the ventricular tissue, indicating that it had left ventricular identity, not right ventricular identity, consistent with results obtained with the tbx5 probe. Tbx20 is highly expressed in the outflow tract and in the A/V canal (Carson et al., 2000; Kraus et al., 2001). Hearts from islet1 mutants expressed tbx20 at the junction of the ventricle and atria, but did not express tbx20 at their anterior end, indicating an absence of the outflow tract. Results consistent with this were obtained with a probe for msx2, which marks the outflow tract of the heart at ED8.5. Islet1 mutant hearts had no anterior staining of msx2. Together with the previous results from hybridization with probes for MLC2a and MLC2v, these data indicated that islet1 mutants were lacking an outflow tract and right ventricle, although cells with left ventricular, A/V canal, and atrial identities were present.
From this analysis, it was inferred that islet1 mutants were missing complete segments of the heart. Additionally, mutant hearts had not undergone looping. This conclusion was strengthened by scanning electron microscopy analysis. Intriguingly, cardiac primordia in isl 1 mutants at ED 9.0 (12 somite pairs) resembled cardiac primordia seen in wild type embryos at earlier stages, at ED 8.25 (5 somite pairs) (Kaufman, 1999), indicating an interruption in heart development. A comparison of wild type littermates to their mutant counterparts at ED 9.5 (22 somite pairs) showed an absence of outflow tract and right ventricle in mutants, consistent with marker analysis.
The severe cardiac phenotype of isl 1 −/− mice led to investigation of expression of isl 1 during early stages of mouse heart development. Single and double whole mount in situ hybridization was performed on embryos from ED7.25 to ED 8.75, utilizing probes for isl 1 and MLC2a mRNAs. The latter is one of the earliest markers for differentiated cardiogenic precursors. Results of this whole mount in situ and subsequent section analysis demonstrated that islet1 is never co-expressed with MLC2a, but rather is expressed in an immediately adjacent population of cells. At the early cardiogenic crescent stages, islet1 expressing cells are medial and dorsal to MLC2a expressing cells. As the heart tube forms, islet1 positive cells within splanchnic mesenchyme comprising the mesocardium and adjacent to foregut endoderm are contiguous with MLC2a positive cells throughout their extent, including anterior and posterior regions. Islet1 is expressed in both splanchnic mesoderm and in ventral foregut endoderm.
Although islet1 was not expressed in differentiating MLC2a positive myocardial precursors, it was expressed in the region of the recently identified secondary or anterior heart field, that is, splanchnic mesodermal cells of the pharyngeal region. Recent evidence has indicated that the anterior heart field in mouse contributes to the outflow tract (Kelly and Buckingham, 2002). This observation, in concert with the cardiac phenotype in islet1 mutants, indicated that islet1 expressing cells might contribute to the outflow tract of the heart.
To investigate this question, lineage analysis of isl 1 expressing cells was performed, by crossing an islet1-cre mouse (Srinivas et al., 2001) with the Rosa26-lacZ indicator mouse (Soriano, 1999). In progeny of this cross, Cre-mediated excision brings the lacZ gene under the control of the ubiquitously expressed Rosa26 locus, enabling the fate of isl 1 expressing cells to be followed by staining for β-galactosidase activity, even when transcription from the endogenous isl 1 locus has been repressed. Results of this analysis were startling, and demonstrated that cells which previously expressed islet1 make a substantial contribution to the embryonic heart, comprising a majority of cells in the outflow tract, right ventricle, and atria, and also contribute to specific regions of the left ventricle. The β-galactosidase positive cells were also observed within the endocardium, and within endothelial cells lining the aortic arch arteries. The majority of β-galactosidase negative myocardial cells were observed within the ventral aspect of the left ventricle and the anterior ventral region of the left atria.
The observation that islet1-expressing cells contribute a majority of cells to the developing heart was consistent with our previous analysis of the cardiac phenotype in isl 1 homozygous mutant mice, where whole segments of the heart were missing. The missing structures indicated that Islet1 might be required for proliferation, survival and/or migration of islet1 expressing cardiogenic precursors. To address this question, an attempt was made to examine isl 1 expressing cells within isl 1 mutants and littermate controls. Although targeting of the isl 1 gene deleted the third exon, containing the second LIM domain, the 5′ end of isl 1 mRNA is still expressed in the mutant, enabling detection of islet1 message in mutant cells. Islet protein, however, is not detectable (Pfaff et al., 1996).
To track isl 1 expressing cells in mutant and wild type embryos, whole mount in situ hybridization analysis was performed on embryos from ED8.5-ED10 with a probe for isl 1 mRNA. Results of this analysis demonstrated that there are progressively fewer islet-expressing cells in the mutant, although some cells still remain. In conjunction with the cardiac phenotype of isl 1 mutants, these results indicate that Islet is required for cell proliferation and/or cell survival.
The results of these studies show that Islet1 is required cell for proliferation and survival of cardiogenic precursors, and that downstream targets of Islet1 are mediating this effect. Two growth factor pathways which have been implicated in growth and survival of cardiogenic precursors in both vertebrate and invertebrate heart development are bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs) (Kirby, 2002; Yutzey and Kirby, 2002). A number of BMPs and FGFs have been described as being expressed in embryonic regions that overlap with and/or are adjacent to islet1-expressing cells, including BMPs 2, 4, 6 and 7, and FGFs 4, 8, and 10 (Crossley and Martin, 1995; Dudley and Robertson, 1997; Kelly et al., 2001; Lyons et al., 1995; Niswander and Martin, 1992). To determine if any of these are targets of Islet1 action, whole mount in situ hybridization was performed with probes for these growth factor genes. Results of this analysis demonstrated a decrease in expression in each of these genes in isl 1 null mice. Expression of some of these growth factors was severely downregulated or undetectable in regions that overlapped islet1 expression, including expression of BMP4, BMP7, and FGF10. These genes are likely to be direct or indirect targets of Islet. Expression of the other BMP and FGF genes was still present, but the domain of expression was decreased in regions overlapping with islet1 expression, similar to results observed with islet1 mRNA in islet1 mutants. This decrease may reflect a decrease in the number of cells that express these growth factors.
The data described herein demonstrate that progenitors which give rise to the outflow tract also give rise to a majority of cells in both the right ventricle and the atria, and a subset of cells within the left ventricle. Thus, the previously described secondary or anterior heart field is a subset of this progenitor population, which is marked by islet1 expression. Islet1 function is required for these cells to contribute to the heart. In the absence of Islet1, hearts which form are missing segments normally contributed by islet1 expressing progenitors. In distinction, progenitors that will give rise to the majority of cells of the left ventricle and a subset of atrial cells do not express islet1, and are capable of giving rise to cardiac cells of these identities in the absence of Islet1 function.
The appearance and characteristics of the heart in islet1 mutants, and the analysis of islet1 expression and fate mapping of islet1 progenitors, have led to a new working model of heart development. In this model, the first protruding segments of cardiogenic mesoderm at the midline are the first to differentiate, do not express islet1, and will give rise to a majority of cells within the left ventricle and some of the adjacent atrium. Islet1 expressing progenitors migrate in, progressively differentiating and downregulating islet1 expression as they join the “primary” heart segments, to form the majority of cells of the right ventricle, outflow tract, and remainder of the atrium. It should be noted that a substantial number of descendents of islet1 expressing progenitors were found within the left ventricle, at the junctional region with the right ventricle, within trabeculae, and along the wall of the inner curvature, descending slightly into the dorsal wall of the left ventricle.
During earliest stages of heart development, islet cells migrate in throughout the anterior-posterior extent of the myocardium, when adjacent mesenchyme is contiguous with differentiating myocardium. At later stages, islet progenitors migrate into the heart through the two regions which remain connected to the splanchnic mesenchyme of the dorsal body wall. Anteriorly, this is the region of the aortic sac and posteriorly, the dorsal mesocardium.
Previous anatomical analysis of human heart development utilizing molecular markers has indicated that extra-cardiac mesenchyme, which migrates in through the dorsal mesocardium, contributes to both atrial and atrio-ventricular septation (Lamers and Moorman, 2002). There is controversy as to whether the mesenchymal cap on the leading edge of the primary atrial septem originates from this extra-cardiac mesoderm, or derives from cushion endothelium. This question can now be definitively addressed by islet1 lineage analysis. Furthermore, it will be of interest to investigate the role of islet-derived myocardial cells in cardiac septation generally.
It is interesting to note that descendents of islet-expressing progenitors markedly populate regions that coincide with markers of the developing conduction system, indicating that this population may play a major role in conduction system development (Rentschler et al, 2002; Kondo et al, 2003).
The data described herein demonstrate that in the absence of Islet1, islet1 expressing cardiac progenitors do not substantially contribute to the heart, and are decreased in number, demonstrating that Islet1 is required for proliferation and/or survival of these progenitors. The observation that most descendents of islet1 progenitors are not present in hearts of isl 1 −/− mutants indicates that Islet1 may also be required for migration.
Islet1 expression is down regulated as precursors differentiate, indicating that, in cardiogenic precursors, Islet function may be required to maintain an undifferentiated state, and/or may be incompatible with a differentiated state. Islet1 is also required for cell survival in motor neurons, but is expressed and functions in differentiated cells (Pfaff et al., 1996). In pancreatic development, Islet1 function is required in both pancreatic mesenchyme, and in differentiated islet cells (Ahlgren et al., 1997).
Growth of the heart following myocyte differentiation has led to the belief that extensive proliferation of differentiated myocytes occurs, to account for myocardial growth. The migration of islet1 expressing precursors into the heart indicates that some growth of the heart can also be accounted for by this migration. However, in islet1 mutants, non-islet expressing progenitors differentiate and undergo an expansion, indicating that both migration and proliferation of differentiated precursors play a role in cardiac growth.
The hearts of islet1 mutants do not appear to have undergone looping morphogenesis, in that the ventricular segment remains anterior to the atrial segment. This observation indicates that migration of islet1 expressing progenitors into the heart is intimately linked to the process of looping morphogenesis. Looping morphogenesis may be a result, not only of myocardial growth, but also of the migration of islet1-expressing cells into the heart.
In addition to being expressed in cardiogenic mesoderm, islet1 is expressed in pharyngeal endoderm, a tissue that has been demonstrated to play an important role in heart development (Lough et al,). This raises the possibility that the requirement for islet in cardiac progenitors may not be cell autonomous. Future experiments will be directed toward this question.
The studies described herein demonstrate that Islet1 defines and is required for one of the cardiogenic fields. It is of interest to understand other genes that may be similarly required for the other, non-islet expressing field. In this context, it should be noted that the Nkx2.5 knockout mouse has a mutant heart that has an outflow tract, right ventricular cells, and atrial cells (Harvey et al, 1999; Tanaka et al, 1999). A number of markers for left ventricular identity are absent, indicating absence of left ventricular identity. These observations raise the possibility that Nkx2.5 is required for formation of cardiac tissue from non-islet expressing progenitors. Nkx2.5 may also play a role in the islet-expressing heart field, although it clearly is not required in the manner that Islet is, given the contrasting phenotypes of islet1 and Nkx2.5 null mice. Creation of mice that are doubly mutant for islet1 and Nkx2.5 can be used to assess these possibilities. It has been shown that islet1 mRNA expression is maintained in Nkx2.5 knockout mice (unpublished observations).
As discussed above, Islet1 positive progenitors may influence cardiac looping morphogenesis. Looping of the heart occurs in a characteristic left-right orientation, with the outflow tract and right ventricle looping rightward. Perturbation of left-right axis information can result in situs inversus of the heart, a leftward looping of the outflow tract and right ventricle. Atrial isomerism can also result. The data described herein demonstrate that the outflow tract, right ventricle, and a majority of atrial cells derive from islet1-expressing progenitors, indicating that left-right information imparted to these precursors will be a critical determinant of left right cardiac asymmetry. Previous analysis of genetic markers has indicated that initial left-right axis information is preserved in the arrangement of the atria, but is rotated in the ventricles. That is, that the “left” and “right” ventricles do not strictly correspond to the left and right body axis (Campione et al., 2001; Franco et al., 2000). Our findings that the left and right ventricles derive from distinct cardiogenic fields gives further insight into this observation. It will be of interest to re-examine left right patterning of the heart in light of the islet1 progenitor population, to investigate genes involved in imparting left right information to these cells prior to their entering the heart, and their subsequent positioning within the heart relative to their original left-right orientation. Similarly, it will be of interest to examine left right identities imparted to non-islet expressing progenitors, and the final positioning of left and right segments within the developed heart.
A homeodomain transcription factor, pitx2, is downstream in the left-right pathway, is specifically expressed in left lateral mesoderm, and remains regionalized with later development (Capdevila et al, 2000). Recent analysis has demonstrated that pitx2 is asymmetrically expressed in the region of the cardiogenic crescent, and later is expressed in left, not right atria, and in distinct portions of the outflow tract, right and left ventricles (Campione et al, 2001). It will be of great interest to investigate potential asymmetric expression of pitx2 in islet-expressing progenitors and perhaps use pitx2 expression as a marker to investigate migratory paths undertaken by islet1-expressing progenitors. In this regard, at ED9.5, pitx2c is expressed asymmetrically in branchial arch and splanchnic mesoderm adjacent to the aortic sac (Liu et al, 2002). Pitx2 knockout mice are nonviable, and display a number of cardiac phenotypes, although hearts still apparently loop to the right (Capdevila et al, 2000). It will be intriguing to re-examine the pitx2 null phenotype, and other issues of left right cardiac asymmetry, within the context of the islet1 paradigm.
Absence of Islet resulted in reduction of the number of islet1 expressing cardiogenic precursors, indicating that growth factor signalling may be perturbed. As both FGF and BMP signalling are required for cardiogenesis (Kirby, 2002; Yutzey and Kirby, 2002), we examined expression of a number of BMP or FGF growth factors which are expressed within or adjacent to islet1 expressing cardiac progenitors. The data described herein demonstrated a significant downregulation or decrease in each growth factor examined, selectively in regions which overlap with islet1 expressing cardiogenic precursors.
Islet1 mutants exhibited an overall reduction in the domain of fgf8 expression, but their phenotype was more severe than that seen with fgf8 hypomorphs. Mouse knockouts of fgf4 or fgf8 are early embryonic lethal, but mice which are hypomorphic for fgf8 die neonatally due to cardiovascular defects, including malformations of the outflow tract (Abu-Issa et al., 2002; Feldman et al., 1995; Frank et al., 2002; Sun et al., 1999). In islet1 mutants, fgf10 expression was virtually absent in islet1-expressing cardiogenic precursors. Mouse knockouts of fgf10 die neonatally, which has been ascribed to their lung phenotype (Sekine et al., 1999). However, there may be an as yet undescribed cardiac phenotype, albeit clearly not as severe as the islet cardiac phenotype.
BMP4 and BMP7 are co-expressed in a highly overlapping manner with islet1 expressing cardiogenic precursors. BMP2 and BMP6 are expressed in a distinctive manner from BMP4, BMP7, or each other, but their expression also overlaps with that of islet1. In islet1 mutants, expression of each of these growth factors was greatly reduced or absent in regions coincident with islet1 expression. Knockouts of each of these BMPs have been made, and double knockouts of BMP6/7 have been made (Kim et al., 2001; Winnier et al., 1995; Zhang and Bradley, 1996). None of these mutants recapitulates the cardiac phenotype of the islet1 mutant, due to earlier defects in implantation or gastrulation, or, if they survive the earlier defects, potentially due to functional redundancy.
Our results indicate that the cardiac phenotype in islet1 mutants may be all or in part due to defects in either FGF or BMP signalling, or both. Discriminating between these possibilities will require future experiments to ablate these signalling pathways in islet1 expressing progenitors. Additionally, other growth factor pathways may be affected in islet1 mutants.
Expression of islet1 in the splanchnic mesoderm of the pharyngeal and foregut region is intriguing in view of a number of experiments which have indicated that the vertebrate heart has evolved from pharyngeal or gut mesoderm (Haun et al., 1998; Park et al., 1998; Ranganayakulu et al., 1998). Previous data has demonstrated that islet1 is expressed in cardiogenic precursors in chick (Yuan and Schoenwolf, 2000). There is a Drosophila homologue of islet which, as for mouse islet1, has a role in motor neuron development, and, intriguingly, is expressed in the dorsal vessel (Thor and Thomas, 1997). It will be of great interest to examine the role of islet in cardiogenesis in other species, to gain further insight into cardiac evolution. Results may indicate that islet-expressing progenitors are, in evolutionary terms, “primary”. If this were the case, it might indicate that the left ventricle was a later evolutionary development. Interestingly, in zebrafish, which have a single ventricle, DHand, a marker of right ventricle in higher vertebrates, is present, whereas no orthologue of its left ventricular counterpart, EHand has been found (Angelo, Lohr, Lee, Ticho, Breitbart, Yost, 2000).
Perhaps one of the most exciting aspects of our discovery of the role of Islet in cardiogenesis is the prospect of utilizing Islet1 as a marker for the cardiogenic stem cell state. A stem cell can be defined as a progenitor cell which can proliferate and give rise to a number of distinct lineages. Islet1-expressing cells conform to this definition, giving rise to a number of distinct cardiac lineages. The unique property of islet1 in being expressed in cells prior to differentiation should allow for cell sorting on the basis of islet1 expression. Additionally, Islet1's role in dictating the proliferation and/or survival of these cells indicate that Islet1, in concert with other factors, may be utilized to drive a cardiogenic stem cell state.
Based on these considerations outlined above, a rare cell population was identified within mouse, rat and human non-myocyte cell cultures, that can be expanded and propagated in vitro. These cells differentiate not only into mesodermal lineages but also neuroectoderm. We are able to show that cells capable of differentiating in vitro to cells of at least two germ layers can be selected from rodent and human hearts. Islet-1 (isl 1), a LIM-homeodomain transcription factor, marks these undifferentiated progenitor cells and allows a visualization of this cardiogenic precursor population in the adult heart. Therefore these cells were termed i-cells.
Culturing of undifferentiated i-cells, as set forth in Example 1 below, revealed that the cardiogenic precursor population could only be cultured without differentiation and senescence on a feeder layer of species-specific cardiac fibroblasts. Similar feeder-dependent culture conditions are used for the isolation of mouse and human ES cells and such feeder layers proved to be critical to maintaining them in an undifferentiated state (Donovan and Gearhart, 2001).
The requirement for feeder cells or conditioned medium from cardiac fibroblasts indicates that they provide factors that suppress the differentiation or promote the self-renewal of the multipotent progenitor cells. An activity with these properties is referred to as differentiation-inhibiting activity of i-cells (DIAI). For murine ES cells, leukaemia inhibitory factor (LIF), a member of the cytokine family related to interleukin-6, can replace the requirement for feeder cells (Nichols, et al., 1990). For inhibiting murine ES cell differentiation activation of the signaling component of the LIF receptor, glycoprotein 130 (gp 130), is both necessary and sufficient. However, human ES cells and cardiac i-cells do not seem to require LIF for blocking differentiation and stimulating self-renewal (Thomson, et al., 1998).
Until now no in vitro culture conditions have been established that allow multipotent, adult stem cells to be expanded and propagated. dult HSCs or NSCs, in vivo defined as long-term repopulating cells, cannot be expanded in culture without losing developmental potential (Weissman, 2000). But one recent study shows that mesenchymal stem cells derived from the bone marrow, can be grown under special conditions indefinitely in culture (Jiang, et al., 2002).
The invention also provides a cellular composition comprising an enriched population of isl 1 positive stem cells. The composition preferably contains a majority of or at least about 90% isl 1 positive stem cells as compared with other cell types. The isl 1 positive stem cells are derived from any cardiac tissue, such as from a rat, mouse or human.
As phenotypic characteristics of an undifferentiated state, it has been discovered that i-cells express high levels of isl 1 in the nucleus and nestin, an intermediate filament that marks undifferentiated NSCs, in the cytosol. Additionally, cells showed lower expression of Nkx2.5, a homeobox vertebrate homologue of Drosophila tinman, and the ES cell transcription factor oct-4. Thus, these markers are useful for identifying i-cells.
Isl 1 is essential for motor neuron and pancreatic development (Pfaff, et al., 1996). Homozygous knock-out embryos for isl 1 die around ED 10.5, because of a univentricular, undivided heart-chamber and vascular abnormalities of the outflow tract (Cai, et al., 2003). Lineage analysis of isl 1 expressing cells revealed that these cells substantially contribute to the embryonic heart, comprising a majority of cells in the outflow tract, right ventricle and both atria (Cai, et al., 2003). The downregulation of isl 1 expression in the neonatal and adult heart allows a visualization of cardiogenic precursors in the myocardium.
Immunohistochemistry showed isl 1 expressing cells in the adult heart mainly localized in the right ventricle, septum and atria. In undifferentiated precursor cells isl 1 is highly expressed in the nucleus and is downregulated during differentiation of i-cells. Islet expression marks a cardiogenic stem cell population in vitro and in vivo and appears to be required for differentiation and survival of these stem cells.
Nkx2.5 is also expressed in i-cells in an undifferentiated state. This transcription factor is one of the earliest markers of vertebrate heart development and is important for the regulation of cardiac-restricted gene activity. The POU-domain transcription factor oct-4 is a molecular marker for pluripotent ES cells. Oct-4 is expressed in the pre-gastrulation embryo, early cleavage-stage embryo, cells of the inner cell mass of the blastocyst and in embryonic carcinoma cells. In the adult animal oct-4 is only found in germ cells and mesenchymal stem cells (Rosner, et al., 1990).
By the present in invention, a novel cardiac progenitor population has been discovered, isolated and characterized. Isl 1 expression marks these cardiogenic stem cells and appears to be required for the differentiation state and survival of these cells. These cells are useful to study and understand signalling pathways of cardiac stem cell differentiation and growth, paving the way for future therapeutic applications for congenital and adult heart disease.
Gene targeting in i-cells with further differentiation in neurons or myocytes allows studies in cell biology without having to struggle with the complexity of time consuming animal models. The aim of cell culture is to develop well defined and easily manipulated experimental systems that offer the advantages of clonal homogeneity and the ability to manipulate external environments. Furthermore, due to the ethical unacceptability of experimentally altering the human germ line, the ES cell transgenic route is not available for experiments that involve manipulation of human genes. Gene targeting in human i-cells allows important applications in areas where rodent model systems do not adequately recapitulate human biology or disease processes.
In addition, I-cells can be useful as a source of donor tissue for cardiac and neuronal cell therapy. Early embryonic stem cells have disadvantages for cell-based therapy: (i) the number of transformations and (ii) the complexity of signals required to achieve a specific differentiated phenotype. Instead, the phenotypic differentiation of developing i-cells and adult-derived cardiac progenitors circumvent both ethical and immunological constraints.
Cross-lineage transformation of i-cells offers a new avenue for a more flexible tissue source, in particular to derive autografts from patients themselves. A further advance of
i-cells compared to ES cells is that these precursor cells are less immunogenic than primary embryonic myocytes in xenografts, highlighting a way to overcome one of the main difficulties of transplantation from non-human donors.
The invention methods utilize isolated monoclonal antibodies characterized as specifically binding to Islet 1 polypeptide and immunoprecipitating the Islet 1 polypeptide
Any suitable immunoassay format known in the art and as described herein can be used to detect the presence of and/or quantify the amount of Islet 1-expressing cells in a diverse population of cells. Although any type of anti-Islet 1 polypeptide antibody, as described herein, which binds specifically to Islet 1 polypeptide, can be used in the invention methods, monoclonal antibodies are preferred.
The invention immunological tests for Islet 1 polypeptide can be used in a high throughput format using any technique known in the art, such as FASC screening as is described below in greater detail.
Detectable labels suitable for binding to antibodies used in the invention methods, including high throughput screening formats, include radiolabels linked to the antibodies using various chemical linking groups or bifunctional peptide linkers. A terminal hydroxyl can be esterified with inorganic acids, e.g., 32 P phosphate, or 14 C organic acids, or else esterified to provide linking groups to the label. Enzymes of interest as detectable labels will primarily be hydrolases, particularly esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and so forth. Chemiluminescers include luciferin, and 2,3-dihydrophthalazinediones (e.g., luminol), and the like.
Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or immunoprecipitation of Islet 1 polypeptide. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene, for example protein G covered wells of microtiter plates or beads.
Antibodies directed against a specific epitope, or combination of epitopes, so as to bind specifically with the Islet 1 polypeptide will allow for the screening of cell populations as described herein. Various screening techniques can be utilized using such monoclonal antibodies, and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
The antibodies useful in the invention methods may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used, include but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., Western blot analysis. Those of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32 P or 125 I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. Those of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. Those of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3 H or 125 I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with the antibody of interest conjugated to a labeled compound (e.g., 3 H or 125 I) in the presence of increasing amounts of an unlabeled second antibody.
Antibodies used in invention assay(s) can be polyclonal, monoclonal, or a functionally active fragment thereof. Mono- or poly-clonal antibodies to a islet 1 polypeptide are raised in appropriate host animals by immunization with immunogenic conjugate(s) using conventional techniques as are known in the art.
The preparation of monoclonal antibodies is disclosed, for example, by Kohler and Milstein, Nature 256:495-7, 1975; and Harlow et al., in: Antibodies: a Laboratory Manual , page 726 (Cold Spring Harbor Pub., 1988), which are hereby incorporated by reference. Briefly, monoclonal antibodies can be obtained by injecting mice, or other small mammals, such as rabbits, with a composition comprising an invention immunogenic conjugate whose preparation is disclosed above, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Barnes et al., Purification of Immunoglobulin G (IgG), in: Methods in Mol. Biol., 10: 79-104, 1992). Antibodies of the present invention may also be derived from subhuman primate antibodies. General techniques for raising antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO 91/11465 (1991) and Losman et al., Int. J. Cancer, 46:310-314, 1990.
It is also possible to use anti-idiotype technology to produce monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the “image” of the epitope bound by the first monoclonal antibody.
The term “antibody” as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab′) 2 , and Fv that are capable of binding islet 1 polypeptide These functional antibody fragments are defined as follows:
(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
(2) Fab′, the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule;
(3) (Fab′) 2 , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′) 2 is a dimer of two Fab′ fragments held together by two disulfide bonds;
(4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and
(5) Single chain antibody (“SCA”), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference). As used in this invention, the term “epitope” means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′) 2 . This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R., Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
Fv fragments comprise an association of V H and V L chains. This association may be noncovalent, as described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise V H and V L chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli . The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426, 1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al., U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry, Methods, 2: 106-10, 1991.
The invention methods use monoclonal antibodies characterized as specifically binding to islet 1 polypeptide, wherein Islet 1 polypeptide retains functional activity.
Hybridoma cell lines producing monoclonal antibodies useful in the invention methods for immunocapture of Islet 1 polypeptide are commercially available.
The following examples are intended to illustrate but not limit the invention.
Example 1
Experimental Procedures
Mouse Mutants
The generation of islet null mutants has been previously described (Pfaff et al, 1996). The knockout construct deleted the exon encoding the second LIM domain of Islet 1. Islet1-cre mice were generously provided by Thomas M. Jessell, and have been previously described (Srinivas et al, 2001). An IRES-cre cassette was inserted into the exon encoding the second LIM domain of Islet1, disrupting islet gene expression.
Whole Mount RNA In Situ Hybridization
Whole mount RNA in situ hybridization was carried out as previously described (Wilkinson, 1999). References for specific RNA probes which were used are as follows: MLC2a (Kubalak et al., 1994); MLC2v (O'Brien et al., 1993); tbx5 (Bruneau et al., 1999); tbx20 (unpublished results); BMP2, BMP6, BMP7 BMP4, BMP5 (Kim et al., 2001; Lyons et al., 1995); FGF4, FGF8 and FGF10 (Feldman et al., 1995; Sun et al., 1999); EHand (Cross et al., 1995); islet1 (EST, GenBank Accession No.: AA198791)(SEQ ID NO:2); msx2 (Liu et al., 1994).
Double RNA in situ hybridization was performed utilizing digoxigenin and fluorescein-labeled probes that were conjugated with alkaline phosphatase (Roche Cat. #1277073, 1685619). Staining reactions were performed with CIP/Ferricyanide/Ferrocyanide according to Janet Rossant's lab protocols website on the worldwide web (mshri.on.ca/rossant/protocols/doubleINsitu) and MagentaPhos-tet Red according to Claudio Stern's lab protocols website (sternlab.anat.ucl.ac.uk/INSITU), or with Fast Red (Roche Cat. No.: 1496549) and BCIP (a chromogenic substrate for alkaline phosphatases) alone. After incubation with and staining to detect the first antibody (Anti-Fluorescein-AP, Roche Cat. No.: 1426338), embryos were incubated at 65° C. for 1 hour to inactivate alkaline phosphatase activity, and washed before incubating with and staining to detect the second antibody (Anti-Digoxigenin-AP, Roche Cat. No.: 1093274). For embryos stained with Fast Red, which is soluble in alcohol, cryosections were prepared. For embryos stained with BCIP/Ferricyanide/Ferrocyanide and MagentaPhos-tet Red, paraffin sections were prepared, with brief washes in alcohol to minimize loss of signal.
Scanning Electron Microscopy
A standardized procedure for scanning electron microscopy (SEM) of the heart was utilized (Pexieder, 1986). Briefly, embryos were submitted to ethanol dehydration and critical point drying from Freon 113 to Freon 23. Dried specimens were mounted on SEM tubes, ion sputtered with 300 nm gold, and examined in the scanning electron microscope. SEM photomicrographs were taken in standard orientations and magnifications.
Example 2
Culture of Undifferentiated I-Cells
To isolate cardiac progenitors from murine hearts, the methods used were similar to those used for cardiomyocyte isolations from the adult organ. In a trypsin-digested state
i-cells and cardiac fibroblasts share a similar cell diameter of around 35 μm and copurify in the same fractions on Percoll gradients. I-cell cultures were developed by testing multiple conditions, including cultures on fibronectin, collagen-type-IV or laminin. Media conditions tested included several concentrations of fetal calf serum (FCS), epidermal growth factor (EGF), platelet derived growth factor (PDGF-BB), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), bone morphogenetic protein (BMP) 2+4, insulin-like growth factor (IGF) 1, sonic hedgehoc (Shh) and dexamethasone, as set forth below.
Approximately 5-10% of 96-well plates seeded with 10 isl 1 positive cells yielded continuous growing cultures, indicating that only around 5-10 cells out of 100 i-cells are capable of initiating i-cell cultures. Several i-cell populations have now been cultured for more than 12 population doublings. It was discovered that the cardiogenic precursor population can only be cultured without differentiation or cell death on a feeder layer of cardiac fibroblasts or in conditioned medium of freshly isolated fibroblasts from the heart. Differentiation of I-cells into myocytes and neurons can be induced by withdrawal of conditioned medium from cardiac fibroblasts or separation of I-cells from the feeder layer. The morphology and phenotype were similar after 5 to more than 10 population doublings.
I-cells were ˜35 μm in diameter with a large nucleus and a scant cytoplasm growing in three-dimensional spheres always attached to the fibroblastic feeder layer. Similar results were obtained when i-cells from atria of human tissue were isolated and cultured.
Example 3
In vitro Differentiation of Single I-Cells
Next in vitro differentiation capacity of i-cells obtained from mouse and rat hearts was tested by adding cytokines chosen on the basis of what has been reported for ES cell differentiation to neuroectoderm and mesoderm. Differentiation required that i-cells had to be replated without a feeder layer at a density around 1-2×10 4 cells cm −2 in medium containing no serum, but lineage-specific cytokines. Neuroprogenitors can be expanded with PDGF-BB and induced to differentiate by addition of bFGF (Palmer, et al., 1999).
Under the bFGF treatment around 45% of the i-cells acquired morphologic and phenotypic characteristics of astrocytes with immunohistochemical positivity for glial acidic fibrillary protein (GFAP) and neurons which stained positive for neurofilament 200 (NF-200). Myocytic differentiation acquired cells that showed positive signals for α-sarcomeric actin and α-actinin in immunohistochemical experiments.
Example 4
Isolation Protocol for Postnatal Cardiac Progenitor Cells from Mouse Hearts
35 to 50 hearts of 1 day old mouse pups were dissected out of the thoraxaperture, cut into four pieces and washed 3× in HBSS (Hank's balanced salt) solution without Ca 2+ at 4° C. The hearts were transferred into 0.5 mg/ml trypsin-HBSS solution and predigested overnight (˜17 hours) at 4° C. on an orbital shaker. Half of the trypsin solution was removed from the predigested tissue and the remaining part diluted 1:1 with warm DMEM/M199 cell culture medium (4:1 ratio) containing penicillin (100 U/ml)/streptomycin (100 mg/ml)/HEPES (25 mM)/glutamine (2 mM).
After shaking the tissue for 3-4 min at 37° C. the diluted trypsin was removed from the tissue and 20 ml of 24 U/ml collagenase type II in HBSS added to the tissue solution. After an incubation of 2 min in a shaking water bath at 37° C., the initial collagenase type II digest is discarded, as it mainly contains red blood cells and dead tissue cells. The tissue was resuspended in 12 ml of fresh collagenase type II and shaken for 10 min in a water bath at 37° C. The supernatant was inactivated by addition of the same volume of cold DMEM/M199 medium containing 10% horse serum and 5% fetal bovine serum and stored on ice. The resuspension of the tissue and the inactivation of the supernatant was repeated three more times until the tissue pieces were completely digested. The supernatants from the digests were pulled together and centrifugated at 800 rpm for 5 min. The supernatant contained most of the mesenchymal cells of the heart and the pellet most of the cardiac myocytes.
After a second centrifugation at 1500 rpm for 3 min, the mesenchymal cells were sequentially plated for 20 min on plastic in DMEM containing penicillin (100 U/ml)/streptomycin (100 mg/ml)/HEPES (25 mM)/glutamine (2 mM)/10% new born calf serum and 5% fetal bovine serum. After 20 min, the non-attached cells were removed from the plates by two rigorous washing steps with PBS.
The attached cardiac mesenchymal cells were cultured for 14-21 days at 37° C. with 5% CO 2 . Culture medium was changed to DMEM/F12 containing B27 supplement, 2% fetal bovine serum, 10 ng/ml EGF at the second day in culture when the cells reached confluency. After 10 days the cardiac progenitor population started to propagate on top of the feeder layer of the mesenchymal cells of the heart.
Example 5
Isolation Protocol for Postnatal Cardiac Progenitor Cells from Rat Hearts
50 hearts of postnatal day 1-5 rat pups were dissected out of the thorax, cut into four pieces and washed 2× in the ADS buffer containing 6.8 g/l NaCl, 4.7 g/l HEPES, 0.12 g/l NaH 2 PO 4 , 0.14 g/l NaH 2 PO 4 H 2 O, 1 g/l glucose, 0.4 g/l KCl, 0.2 g/l MgSO 4 7H 2 O (pH adjustment to 7.35). The heart pieces were incubated for 15 min at 37° C. in ADS buffer containing collagenase type II (115 units/ml) and pancreatin (0.8 mg/ml) in a stir flask. The first digest was discarded. 18 ml of fresh enzyme solution were added to the tissue and stirred for 20 min at 37° C.
After the 20 min digestion, the enzyme solution was removed and inactivated with 6 ml of neonatal calf serum. Fresh enzyme solution was added to the tissue pieces in the stirring flask and incubated for another 20 min at 37° C. The digest from the first 20 minute digestion was centrifuged at 1000 rpm for 6 min, the pellet resuspended in 5 ml of neonatal calf serum and placed at 37° C. in 10% CO 2 . The above steps, from removal of the enzyme solution to resuspension of the pellet was repeated four times.
The resulting cell suspensions from each centrifugation were pooled and the pool was centrifuged at 1000 rpm for 6 min. The pellet was resuspended in 12 ml of ADS buffer. The cell suspension was layered on the top of a Percoll gradient (2 ml cells per each gradient). Each Percoll gradient consists of 4 ml Percoll 1.06 g/ml as the top layer and 3 ml Percoll 1.08 g/ml as the bottom layer.
After centrifugation at 3000 rpm for 30 min with low acceleration and deceleration, the upper band consisted of the cardiac mesenchymal cells, the middle band at the interphase consisted of the cardiac myocytes. The mesenchymal cells were collected with a Pasteur pipette.
After a second centrifugation at 1500 rpm for 3 min, the mesenchymal cells were sequentially plated for 20 min on plastic in DMEM containing penicillin (100 U/ml)/streptomycin (100 mg/ml)/HEPES (25 mM)/glutamine (2 mM)/10% new born calf serum and 5% fetal bovine serum. 20 min later the non-attached cells were removed from the plates by two rigorous washing steps with PBS.
The cardiac mesenchymal cells were cultured for 14-21 days at 37° C. with 5% CO 2 . Culture medium was exchanged to DMEM/F12 containing B27 supplement, 2% fetal bovine serum, 10 ng/ml EGF at the second day in culture when the cells reached confluency. After 10 days, the cardiac progenitor population started to propagate on top of the feeder layer of the mesenchymal cells of the heart.
Example 6
FACS Analysis of Phenotypic Cell Markers of the Cardiac Progenitor Cell Population
The phenotypic cell markers of the isolated cells from postnatal mouse and rat myocardium in Example 4 and Example 5 above, respectively, were characterized by FACS analysis. The FACS analysis showed that: 90% of the cells express the LIM homeodomain transcription factor islet1; ˜90% of the cells coexpress the Drosophila tinman homologue Nkx2.5; and 30-40% of the cells coexpress the intermediary filament nestin.
Example 7
Differentiation Protocols
For differentiation of the progenitor population, the cells were replated without the feeder layer of mesenchymal cells at a density around 2×10 4 cells per cm −2 in a medium containing 2% of fetal calf serum and the lineage-specific differentiating agents.
In vitro myocytic differentiation was performed with conditioned medium enriched for wnt11 of a retrovirally infected NIH3T3 cell line, which stably expresses and secretes wnt11. Cells were treated with a sequential differentiation protocol for 4.5 days with wnt11 conditioned medium on fibronectin coated culture dishes and afterwards with BMP2 in a concentration of 2.5 ng/ml. Thereafter differentiated cells were analyzed in single cell experiments for channel currents in an electrophysiological setting and for intracellular Ca 2+ transients.
In vitro differentiation in neuronal cell types was performed with 0.2 μM all-trans retinoic acid and 5 μM forskolin for 10-15 days in laminin and polylysin coated culture dishes.
In vitro differentiation in adipocytes was performed with 10% neonatal calf serum and 5% fetal bovine serum in plastic culture dishes.
Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.
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