Patent Application
20080280295
The present invention relates to a panel comprising at least two pairs of primers that are complementary to at least two different reporter genes, wherein the expression of the at least two reporter genes are
i) either up- or down-regulated upon cell differentiation, and
ii) display a similar expression profile in at least two different cell lines of the same kind of cells.
The panel is suitable for use e.g. in a method for rapid and sensitive determination of the state of differentiation of e.g. hBS cells. The method is based on the analysis of mRNA levels of specifically selected genes using quantitative real time PCR (QPCR).
The successful isolation and culturing of human embryonic stem cells in 1998 marks the birth of a new era in biomedical research with wide-ranging implications for basic research, drug development and the treatment of disease. The worldwide interest in the potential of human blastocyst-derived stem cells or hBS cells is based on their unique ability to form all specific cell types and tissues found in the human body. A further important characteristic of stem cells is their capacity to reproduce themselves continuously under certain culture conditions, known as self-renewal.
Adult stem cells have been used previously for transplantation in a number of different applications, such as blood transfusion and in bone marrow, cartilage, skin and cornea transplants. However, these cells are limited in their potential to differentiate into other cell types. On the other hand, the hBS cells are pluripotent, which means that they have the potential to develop into any of the tissues and organs found in the body. However, the hBS cells are not totipotent. It is anticipated that the unique properties of hBS cells will make these cells useful in several different applications such as:
To generate new hBS cell lines, donated fertilized oocytes produced by in vitro fertilization for clinical purposes are used and grown to the blastocyst stage. Cells when employed according to the invention are derived from the blastocyst 4-5 days after fertilization and are therefore referred to as blastocyst-derived stem cells or hBS cells. The inner cell mass of the blastocyst is isolated by various methods including micro- or immunosurgery and plated on mitotically inactivated murine embryonic fibroblasts in a specific culture media. Under defined culture conditions undifferentiated hBS cells can be propagated indefinitely in vitro. The hBS cells can also undergo spontaneous differentiation and it is important to monitor the hBS cell culture over time. Various markers and assays can be employed to monitor hBS cells and undifferentiated hBS cells can be identified based on the following characteristics:
Visual inspection of the morphology of hBS cells in culture is a rapid but subjective characterization method, requiring extensive experience, by which it is possible to partly distinguish between undifferentiated and differentiated cells. The typical morphology of an undifferentiated hBS cell colony is recognized by tightly packed round shaped cells with a high nucleus to cytoplasm ratio.
The expression of different surface antigens (SSEA-1, -3, -4, TRA-1-60 and -1-81) is in most cases analyzed by immunohistochemistry, which gives a good indication of the phenotype of the cells although accurate quantitative data are difficult to obtain. A second drawback is that the antigens might be expressed at the cell surface even after the initiation of differentiation of the hBS cells (due to a slow turnover).
Oct-4 is a transcription factor expressed by undifferentiated hBS cells and it is known to be down-regulated upon differentiation into different precursor cells. Oct-4 can be analyzed either on the protein level (using for example immunohistochemistry or westen blot) or on the level of gene expression. However, it should be noted that Oct-4 is not exclusively expressed by undifferentiated hBS cells.
It has been shown that hBS cells display a high activity of alkaline phosphatase, but it is also known that this enzyme is also active during differentiation of hBS cells and in other cell types.
By cytogenic analysis of hBS cells, chromosomal abnormalities in the cells can be detected, which gives valuable information about the genetic integrity of the cells but it provides no indication of the state of differentiation of the cells.
High telomerase activity in cells indicates their ability to proliferate, which is a key characteristic of hBS cells. Still, some variations in telomerase activity within and between different hBS cell lines have been reported and it remains to be determined if there is a direct quantitative relationship between telomerase activity and differentiation state of the hBS cells.
Benign teratomas can be generated by injection of hBS cells into immuno-compromised experimental animals. If cells from all three embryonic germ layers are represented within the teratoma, this demonstrates the pluripotency of the injected hBS cells. This method is very time consuming and requires animal experiment, but apart from this, it is an excellent functional test of hBS cells. However, quantitative data are difficult to obtain.
Similar verification of the pluripotency of hBS cell can be performed in vitro. For such studies, hBS cell are induced to differentiate, either spontaneously or in a directed fashion, into various mature cell types representing the three embryonic germ layers. Compared to the in vivo method indicated above, this assay does not involve any animal experiments but it is still time and labour consuming and difficult to evaluate in a quantitative manner.
Genetic characterizations, such as DNA micro arrays have previously been used in order to detect genes that are up- and down regulated during differentiation (US20030224411). Major drawbacks with that technique are the lack of sensitivity and complexity if to applied as a routine analysis.
Taken together, the different characterization methods discussed above, illustrate well the status of hBS cells. However, the individual analyses each have drawbacks such as not being quantitative, requiring animal experiment, not being exclusively specific for hBS cells and some are not possible to perform in large scale. In addition, to perform all the characterization analyses mentioned above is very time consuming. Therefore, there is a need for new, simple, fast and reliable methods for the rapid and quantitative identification and characterization of hBS cells.
The expression of certain genes can be analyzed using QPCR, a sensitive method for detecting mRNA species in biological samples. The specificity of the assay depends to a large extent on the design of specific primers and other reaction conditions that are necessary for the PCR-reaction to proceed. With PCR it is possible to detect a single copy of template because of the exponential amplification during temperature cycling. A single copy of template will result in billions of copies after just 20 cycles (220), which can be readily detected using standard techniques. Traditionally, the obtained PCR products are detected using intercalator dyes such as ethidium bromide after separation on agarose gel by electrophoresis. Semi-quantitative information can be obtained by comparing intensity of bands on the gel, but since the amplification in many cases has entered into plateau-phase comparison between samples is difficult and inaccurate. The need to analyse all PCR products by gel electrophoresis also increases the risk of contamination when post-PCR tubes are opened.
In QPCR a fluorescent dye is included in the PCR reaction and the fluorescent signal is monitored throughout the amplification. This way one can compare how many rounds (cycles) of amplification are necessary to reach a detectable signal and is indicatory of the initial amount of template in the sample. This signal level (threshold level) is always set in the exponential phase of amplification where samples can be directly compared. Traditionally, a target gene of interest has been compared to some kind of reference gene, to take into account differences in extraction yields, reverse transcription efficiencies and differences caused by inhibitors within the samples. The difficulty has been to find suitable reference genes with stable expression throughout the study intended. In many cases housekeeping genes such as GAPDH, β-tubulin and β-actin have been chosen, but it has been demonstrated that these genes are also regulated in many situations and can therefore cause erroneous results.
As mentioned above, there is a need for developing complementary and more specific methods for identification of undifferentiated and differentiated hBS cells. The increasing research and development within the stem cell field also requires suitable and reliable methods for hBS cell characterization that can be used in both small and large scale. Such method can be applied for various purposes, such as:
Despite a wide spread use of many hBS cell markers (discussed above) there is a general lack of information related to how well these markers and methods actually reflect the undifferentiated and pluripotent state of hBS cells.
The present inventors have found that all known reporter genes are not equally suited for use as markers for determining the differentiation step of hBS cells. In particular, the inventors have found that it is advantageous to evaluate the expression level of a combination of reporter genes, each of which should have a similar expression profile in at least two different cell lines of the same kind of cells.
Accordingly, in one aspect the invention relates to a panel comprising at least two pairs of primers that are complementary to at least two different reporter genes, wherein the expression of the at least two reporter genes are
i) either up- or down-regulated upon cell differentiation, and
ii) display a similar expression profile in at least two different cell lines of the same kind of cells.
In the following reporter genes having the above-mentioned properties are denoted “stable reporter genes”.
Preferably, the cells are BS cells such as hBS cells, and/or the cell line is a BS cell line such as a hBS cell line.
As used herein, the term “panel” refers to a combination of pairs of primers, each pair being complementary to a reporter gene, the expression of which is to be investigated.
As used herein the term “up-regulated” is referring to a positive statistically significant (+/−2SD) difference in gene expression. From the examples herein it is seen that e.g. AFP (alpha-feto-protein) is an example of a reporter gene, the expression of which is up-regulated.
In a similar manner, the term “down-regulated” is referring to a negative statistically significant (+/−2SD) difference in gene expression. As seen from the examples herein e.g. Oct-4, Nanog and Cripto are reporter genes, the expression of which are down-regulated.
As used herein the term “expression profile” is referring to the expression of one or more specific genes either constantly increasing or decreasing within a certain amount of time.
The present inventors have observed that even if the expression of some reporter genes are up- or down-regulated in a certain cell line this is not necessarily a stable feature, e.g., if the expression of a reporter gene is up-regulated in a certain cell line, then it cannot be expected that the expression of the same reporter gene is up-regulated in another cell line of the same type of cells. However, with respect to the expression of certain specific reporter genes, the present inventors have found that these reporter genes behave in the same manner irrespective of the cell line (provided that it is the same kind of cells). More specifically, the inventors have found that this applies for at least two, preferably at least three, at least four, at least five or at least six cell lines, cf. the examples herein.
More specifically, the present inventors have found that the expression of the reporter genes encoding Cripto, AFP, Oct-4 and Nanog are either up- or down-regulated upon differentiation and display similar expression profiles between cell lines of the same kind of cells. The expression of other reporter genes investigated did not comply with both of these criteria.
The present invention is based on these results that provide new possibilities with respect to how to e.g. evaluate and determine e.g. the differentiation state of a cell population.
Furthermore, the present invention provides inter alia
i) methods for analysing different genes, known to be expressed in undifferentiated hBS cells. Taken together, the expression levels of these genes provide valuable information about the status of the hBS cells,
ii) methods for analysing the inclusion of genes specifically expressed by differentiated hBS cells. The combination of genes expressed in both undifferentiated and differentiated hBS cells makes the method extremely sensitive and accurate; an important advantage of combining these kinds of genes is the exclusion of traditional reference genes, since many of these are known to be regulated as well and can therefore cause erroneous results,
iii) use of a mathematical model, which is based on the ratio of geometric averages of the multiple genes analysed. With this model it is possible to determine an expression index based on the relative mRNA levels two or more key genes in a biological sample.
iv) use of the expression indices calculated to compare the differentiation state of different populations of hBS cells.
With the methods described herein using the knowledge of specific reporter genes, it is possible to distinguish, with high resolution, between undifferentiated and differentiated hBS cells. Undifferentiated hBS cells will result in a high expression index, whereas differentiated hBS cells will result in a low expression index.
In the methods described herein a panel according to the invention is used. In a specific embodiment, the panel is used in assays for real-time PCR analysis.
Depending on the specific use of the panel, a panel according to the invention may be
i) a panel, wherein the expression of one of the at least two reporter genes is up-regulated upon differentiation of cells,
ii) a panel, wherein the expression of one of the at least two reporter gene is down-regulated upon differentiation of cells,
iii) a panel, wherein one of the at least two pairs of primers is complementary to a reporter gene, the expression of which is up-regulated, and another of the at least two pair of primers is complementary to a reporter gene the expression of which is down-regulated,
iv) a panel, which comprises three or more pairs of primers, which are complementary to three or more reporter genes, wherein the expression of the three or more reporter genes are
i) either up- or down-regulated upon cell differentiation, and
ii) display a similar expression profile in at least two different cell lines of the same kind of cells,
vi) a panel, which comprises four or more pairs of primers, which are complementary to four or more reporter genes, wherein the expression of the four or more reporter genes are
i) either up- or down-regulated upon cell differentiation, and
ii) display a similar expression profile in at least two different cell lines of the same kind of cells.
vi) a panel, which comprises at least two pairs of primers, which are complementary to at least two reporter genes, wherein the expression profile of the at least two reporter genes, particularly when viewed at the respective expression indices as calculated for individual cell lines, is useful to distinguish hBS cells based on their differentiation state.
More specifically, a panel according to the invention is a panel, wherein the at least two pairs of primers are selected from pairs of primers that are complementary to two or more of the reporter genes AFP, Cripto, Oct-4 and Nanog. For example one of the at least two pairs of primers, such as, e.g. three pairs of primers or four pairs of primers, may be complementary to AFP whereas one or more of the other at least two pairs of primers is complementary to a reporter gene, the expression of which is down-regulated upon cell differentiation. Such reporter gene, the expression of which is down-regulated upon cell differentiation, may be selected from the group consisting of Cripto, Oct-4 and Nanog. In another embodiment one or more of the at least two pairs of primers, such as, e.g., three pairs of primers or four pairs of primers, is complementary to a reporter gene selected from the group consisting of Cripto, Oct-4 and Nanog whereas another of the at least two pairs of primers is complementary to a reporter gene, the expression of which is up-regulated upon cell differentiation.
In a specific embodiment of the present invention, the panel comprises two pairs of primers that are complementary to two reporter genes, the expression of which are up-regulated and down-regulated upon cell differentiation, respectively. These two pairs of primers may be complementary to the reporter genes AFP and Cripto, respectively.
The present invention relates to a method of analyzing in concert the expression of two or more different genes in hBS cells, using QPCR, comprising the following steps:
i. Selection of stable reporter genes
ii. Primer design
iii. QPCR
iv. Quantification
v. Comparison
Genes suitable as markers for undifferentiated hBS cells or their differentiated progenies can be selected by studying genetic profiles resulting from e.g. macro scale analysis and literature in the field, such as relevant public or private expression libraries. Results from PCR analysis (see Ct-values) further show that some genes are better suited as reporter genes than others. A reporter gene should have the following basic criteria: a) It should be either up or down regulated upon differentiation of the hBS cells and b) it should display a similar expression profile in several (e.g. more than two different hBS cell lines). Furthermore, when selecting a panel of reporter genes it is of importance that the combination of genes is chosen appropriately in order to maximize the sensitivity of the assay. Here we show that the combination of Cripto and AFP is clearly sufficient to discriminate between undifferentiated hBS cells and early derivatives thereof as further described in Example 9 and 10. Other combinations of potential reporter genes may not provide the same sensitivity. In addition, by adding several appropriate reporter genes (such as Oct-4 and Nanog) further robustness can be added to the assay. On the other hand, if such reporter genes are selected arbitrarily without prior testing there is a significant risk that the sensitivity and robustness of the assay is compromised. The combination of genes in the present invention includes, but is not limited to Oct-4, Nanog, Cripto, and AFP.
A panel could comprise primers complementary to other genes as those listed below, provided that they are
i) either up- or down-regulated upon cell differentiation, and
ii) display a similar expression profile in at least two different cell lines of the same kind of cells.
Examples of genes known to be down-regulated upon cell differentiation and as such may be used as reporter genes according to the present invention include the following genes: Tert (telomerase reverse transcriptase), Sox-2 (homeobox-domain transcription factor 2), Rex-1 (a zinc finger protein), Utf-1 (a transcriptional co-activator), Dppa-5 (developmental-pluripotency associated 5), Cx-43 and Cx-45 (gap junction proteins connexin-43 and -45), ABCG-2.
Examples of genes known to be up-regulated upon cell differentiation and as such may be used as reporter genes according to the present invention include the following genes: the early endodermal markers Brachyury, Sox-17, Goosecoid, Mixl-1, Foxa-2 (sometimes also referred to as HNF-3beta), and CXCR-4, the early mesodermal markers Brachyury (mesendodermal), Meox-1, Fox-F1, KDR, and Bmp-4 and early ectodermal markers such as Sox-1 and Zic-1.
The expression of genes in the present invention can be analyzed individually or in all different combinations such as; Oct-4/AFP, Cripto/AFP, Nanog/AFP, Oct-4*Nanog*Cripto/AFP, Oct-4*Cripto/AFP, Nanog*Cripto/AFP, or Oct-4*Nanog/AFP.
Accordingly, one embodiment of the present invention relates to a panel comprising at least two pairs of primers that are complementary to at least two different reporter genes, wherein the expression of one of the at least two reporter genes is up-regulated upon differentiation of cells and/or wherein the expression of one of the at least two reporter genes is down-regulated upon differentiation of cells. In a further embodiment one of the at least two pairs of primers is complementary to a reporter gene, the expression of which is up-regulated and another of the at least two pairs of primers is complementary to a reporter gene, the expression of which is down-regulated.
As mentioned in the above, evaluation of the expression of more than two reporter genes improves the assay for determination of differentiation state disclosed herein, why another embodiment of the present invention relates to a panel comprising three or more, such as, e.g., four or more, pairs of primers, which are complementary to three or more, such as, e.g. four or more, reporter genes, wherein the expression of the three or more reporter genes are
i) either up- or down-regulated upon cell differentiation, and
ii) display a similar expression profile in at least two different cell lines of the same kind of cells.
Furthermore, one embodiment of the present invention relates to a panel comprising at least two reporter genes, each of which displaying a similar expression profile in at least three, such as, e.g., at least four, at least five or at least six different cell lines of the same kind.
It is disclosed herein, that the expression levels of the reporter genes encoding AFP, Cripto, Oct-4 and Nanog are particularly suitable for evaluation of differentiation state of hBS cells. The Ct values obtained by QPCR in five different cell lines indicated consistent either up- or down-regulation and similar expression profiles of these reporter genes from cell line to cell line, which criteria were not met by the Ct values obtained for the reporter genes encoding DNMT3B, Sox-2, Gdf-3 and β-III-tubulin.
Therefore, one embodiment of the present invention relates to a panel of pairs of primers complementary to at least two pairs of primers selected from pairs of primers that are complementary to two or more of the reporter genes AFP, Cripto, Oct-4 and Nanog.
RNA for use in as template in QPCR can be obtained from pluripotent/undifferentiated BS cells, especially hBS cells. These BS cells can be obtained from a BS cell line, especially an hBS cell line. Although the present invention concerns hBS cells it is contemplated that a method according to the invention also can be applicable to other mammalian BS cells or cell types undergoing differentiation, such as any adult stem cells.
A suitable hBS cell line for use to obtain RNA may be established according to the method for establishing an hBS cell line described in WO 03/055992.
The cells may be in any stage of development. The hBS cells may be cultured with or without supporting material including, but not limited to mouse EF cells, human EF cells and extracellular matrices.
Such suitable hBS cells have at least one of the following properties
In one embodiment the hBS cells have all the properties mentioned above.
In another embodiment of the invention the hBS cells are cultured under differentiating conditions. Differentiation could be performed by any method, such as, e.g. spontaneously in high dense cultures, by the addition of growth factors into the culture media, by co-culture with other cells or cell lines.
In one embodiment of the invention the expression of genes was quantified using QPCR. The buffers and reagents used are, but not limited to, as described in “General description of the procedure”. Similar solutions and reagents may be used. QPCR reactions are preferably run in the Rotorgene 3000 (Corbett Research) but can also be run in any type of real time PCR machine. The temperature of the denaturation/activation step is normally 95° C., the denaturation step is normally 95° C., the annealing step is normally 60° C. and the elongation step is normally 72° C. The time period of the denaturation/activation step is normally 3 min, the denaturation step is normally 20 s, the annealing step is normally 20 s and the elongation step is normally 20 s. The total number of cycles is normally 45. It is emphasized that the skilled artisan will know that temperatures, time intervals and number of cycles can be varied.
Accordingly, in one embodiment of the present invention a panel comprising pairs of primers according to the present invention are used in assays for real-time PCR analysis.
Thus, in one embodiment of the present invention QPCR can be used to select and/or validate reporter genes, the expression of which
i) are either up- or down-regulated upon differentiation of cells, and
ii) display a similar expression profile in at least two different cell lines of the same kind cells.
In a specific embodiment of the invention the expression index, I, (also called ratio) is determined by calculating the ratio of expression of different genes in one sample. The mathematical model used is preferably, but not limited to (Eq. 1):
E is the PCR efficiency, Ct is the threshold cycle, and n and (m-n) are the numbers of genes that are down- and up-regulated, respectively, upon differentiation of hESC. The PCR efficiencies can be evaluated from dilution series of purified PCR products. KRS is the relative sensitivity constant that, among other things, accounts for the differences in templates' fragment lengths. It does not affect relative comparisons of samples. This mathematical model is derived from the equation where 2 genes can be compared (Eq. 2):
where N0 is the initial amount of template copies.
In one embodiment of the present invention, the expression of least two reporter genes is evaluated by calculation of the Expression index, I, upon differentiation of hBS cells. The state of differentiation can then be expressed by means of an expression index.
In order to assess variations in state of differentiation between at least two cell lines the expression of at least two reporter genes can be compared for at least two cell lines by obtaining the ratio between their respective expression indexes.
Since evaluation of differentiation state by calculation of the Expression index can be performed using QPCR data for at least two reporter genes of which one is up-regulated and the other is down-regulated, different combinations of suitable reporter genes can be used. Accordingly, in one embodiment the present invention relates to a panel of pairs of primers, one of the at least two pairs of primers is complementary to either the AFP gene, the Cripto gene, the Oct-4 gene or the Nanog gene.
Performance of combinations of two or more reporter genes can be evaluated by obtaining the ratio between the expression index, I, of undifferentiated cells relative to differentiated cells. Accordingly, the performance of combinations of two or more reporter genes is evaluated by obtaining the ratio between the expression index, I, of undifferentiated cells relative to differentiated cells.
In this way it was found that if only including QPCR data for the expression of two reporter genes when calculating the Expression index, I, the best combination is AFP and Cripto. Accordingly, the present invention relates to a panel of primers complementary to the reporter genes AFP and Cripto, respectively. However, in the same way it was found that if to include QPCR data of a third reporter when calculating the Expression index, I, Oct-4 performs better than Nanog. Accordingly, one embodiment of the present invention relates to a panel of three pairs of primers, which are complementary to the reporter genes AFP, Cripto and Oct-4 and another embodiment of the present invention relates to a panel of four pairs of primers, which are complementary to the reporter genes AFP, Cripto, Oct-4 and Nanog.
Gene sequences can be located and analysed and primers being designed and evaluated using any available software by a person skilled in the art. In a specific embodiment of the present invention genes were located and analysed using Ensembl (http:/www.ensembl.org/). Primers were designed using the Primer3 web-based software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and obtained primers evaluated using Netprimer
(http//www.premierbiosoft.com/netprimer/netprlaunch.html) and Beacon Designer 2.1 (Premier Biosoft).
When possible, primers can be designed to span intron-exon boundaries to ensure that genomic DNA is not quantified. Primers can further be designed for appropriate annealing temperatures, such as between 50° C. and 70° C., such as for 60° C. annealing. To ensure specificity, all primer sequences can be searched in databases, such as the NCBI sequence database using a suitable search engine, such as BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).
Accessibility of the obtained primer sequences can be evaluated and resulting amplified sequence analysed using an evaluation tool such as mFold (http://www.bioinfo.rpi.edu/applications/mfold/old/dna/form1.cgi) showing all probable secondary structures at certain conditions. The melting temperature can be analysed using a tool, such as the Oligonucleotide Properties Calculator (http://www.basic.nwu.edu/biotools/oligocalc.html).
The primers used in the present invention includes, but are not limited to:
In one aspect the present invention relates to generate a second ratio between two or more different cell lines or samples of such for a relative comparison based on the individual expression ratios as herein described for a collection of up- and down regulated genes whereby the KRS can be eliminated.
In further aspects, the invention relates to studying gene expression at any state of differentiation between undifferentiated BS cells and fully differentiated cells of endodermal, mesodermal and/or ectodermal origins such as, e.g., hepatocytes, pancreatic cells, gut epithelial cells, cardiomyocytes, chondrocytes, osteocytes, keratinocytes, neurons, astrocytes and oligodendrocytes. The QPCR assay can be adjusted to suit different pathways. One embodiment of the invention relates to a panel of pairs of primers of which at least one pair of primers is complementary to a reporter gene, the expression of which is up-regulated at any state of differentiation between undifferentiated BS cells and fully differentiated cells of endodermal, mesodermal and/or ectodermal origin, such as, e.g., hepatocytes, pancreatic cells, gut epithelial cells, cardiomyocytes, chondrocytes, osteocytes, keratinocytes, neurons, astrocytes and oligociendrocytes.
The most important aspect is to select and validate appropriate reporter genes. The assay of choice can be exemplified by an assay that can be used to screen extrinsic factors such as substances or growth factors for their ability to induce cardiac differentiation of hBS cells. It is clear that similar assays can be designed for other purposes. The reporter genes should have either of the following criteria: a) Down regulated upon differentiation of hBS cells or b) Up regulated upon differentiation of hBS sells to cardiomyocytes. Genes fulfilling either of these criteria can be identified by screening published data or by screening relevant public or private expression libraries. Examples of genes fulfilling criteria a) are Oct-4, Nanog, and CriPto, and examples of genes fulfilling criteria b) are cardiac-specific alpha-cardiac myosin heavy chain (MHC), beta-MHC, sarcomeric myosin, aipha-actinin, and GATA4. The individual QPCR assays for each gene should initially be optimized and the PCR efficiency determined. The usefulness of the potential reporter genes should also be validated through experimental testing using QPCR and control RNA prepared from undifferentiated hBS cells, differentiated hBS cells (at various stages of differentiation), and cardiomyocytes (adult cells or cells differentiated from hBS cells). In order to design a general assay applicable for any hBS cell line it is further important to select reporter genes with expression patterns that are similar between different hBS cell lines. Once the reporter genes have been selected and validated, they can be used in the QPCR assay and the data evaluated using the Mathematical model in order to calculate the expression index, which quantitatively indicates the potency of each tested substance or factor.
Accordingly, one embodiment of the present invention relates to a panel comprising pairs of primers of which at least one is complementary to a reporter gene which is up-regulated upon differentiation of BS derived cells into cardiomyocytes. Such reporter genes can be selected from reporter genes encoding cardiac-specific alpha-cardiac myosin heavy chain (MHC), beta-MHC, sarcomeric myosin, alpha-actinin, or GATA4.
Following the same approach as described above for the cardiac differentiation, assays for determination of differentiation of hBS cells to other cell types can also be performed. The only difference is that the selection of reporter genes based on critera b (see above) should be focused on the particular cell type(s) of interest. For example, genes such as neuron-specific β-III tubulin, NeuN, DoubleCortin, tyrosine hydroxylase, Map 2, NF-L, NH-H, NSE, DBH, GABA, synaptophysin, and serotonin and/or genes expressed in less differentiated neural lineages, such as Pax-6, EMX-2, Musashi can be tested and validated when differentiation of hBS cells towards neural cell types is preferable.
Accordingly, one embodiment of the present invention relates to a panel comprising pairs of primers of which at least one is complementary to a reporter gene which is up-regulated upon differentiation of BS derived cells into neural cells. Such reporter genes can be selected from reporter genes encoding β-III tubulin, NeuN, DoubleCortin, tyrosine hydroxylase, Map 2, NF-L, NH-H, NSE, DBH, GABA, synaptophysin, serotonin, Pax-6, EMX-2, or Musashi.
Furthermore, genes such as AFP, alpha-antitrypsin, liver fatty acid binding protein, cytokeratin 18, and albumin can be tested and validated when differentiation of hBS cells towards hepatocytes is preferable.
Accordingly, one embodiment of the present invention relates to a panel comprising pairs of primers of which at least one is complementary to a reporter gene which is up-regulated upon differentiation of BS derived cells into hepatocytes. Such reporter genes can be selected from reporter genes encoding alpha-feto-protein, alpha-antitrypsin, liver fatty acid binding protein, cytokeratin 18, or albumin.
In other aspects the present invention relates to use for quality control of undifferentiated hBS cells in culture by performing the QPCR method described herein on a regular basis, such as between every 5th and 10th passage on one or more individual cell lines to verify culture stability, detect intrawell line variations over time and/or detect spontaneous differentiation. This can be performed by using the genes described above being up and down regulated during different lineages of induced differentiation.
In still other aspects the present invention relates to detection of differences in expression profiles between different cell lines at the same time points in maybe the same culture lab, i.e. inter-cell line variations or in order to detect cell line specific reactions to e.g. culture conditions such as oxygen stress and/or medium additives, such as growth factors, support matrices and dissociation methods by using the system herein described for induced and spontaneous differentiation.
In still further aspects the invention can be used as an alternative approach to many functional assays based on e.g. enzymatic activities to detect biological processes in the cells other than differentiation, such as metabolism and/or apoptosis. The assay can be designed after identification and validation of suitable reporter genes being up and down regulated during the process of interest. Up-regulated genes in the case of apoptosis could belong to the family of caspases and for metabolism belong to e.g. liver function related genes, such as different cytochromes. Down regulated genes can be any gene(s) showing a down regulated expression in the biological process of interest. Once the reporter genes have been selected and validated, they can be used in the OPCR assay and the data evaluated using the Mathematical model in order to calculate the expression index, which quantitatively indicates the potency of each tested substance or factor. Through experimental testing using QPCR and control RNA prepared from different samples of untreated hBS cells and or hBS derived cells and hBS cells or hBS derived cells treated with chemical compounds at various concentrations and exposure times substance specific expression indices can be generated. To obtain a general assay applicable for any hBS cell line or hBS cell line derivative the selection of reporter genes with similar expression patterns between different hBS cell lines under slightly varied culture conditions is important.
In still further aspects the present invention relates to use of such systems described above in e.g. the drug discovery process to identify differentiation inducing substances or toxic effects e.g. in candidate drugs and/or lead substances potentially on an industrial scale using high-throughput set-ups. Accordingly, a panel of pairs of primers according to the present invention can be used to measure the response of cells upon addition of chemicals, drug candidates and/or drugs e.g. in a read out assay of hBS cell based methods for drug discovery, in a read out assay of hBS cell based methods in toxicity testing.
In still further aspects the present invention relates to detecting toxic effects of chemicals after certain or discreet exposure times and doses and a subsequent analysis based on e.g. the generations of expression indices for e.g. different biological processes as described above.
In still further aspects the present invention relates to detecting either differentiation inducing or differentiation inhibiting effects of known or novel compounds on BS cells, i.e. in vitro developmental assays.
Furthermore, one aspect of the present invention relates to a kit for use in real-time PCR to assess the state of hBS cell differentiation comprising:
i) a panel as defined in any of claims 1-25,
ii) optionally a suitable PCR mix,
iii) optionally one or more component for reverse transcription,
iv) optionally, instructions for use thereof.
As used herein, the term “blastocyst-derived stem cell” is denoted BS cell, and the human form is termed “hBS cells”.
As used herein, the term “embryoid bodies” is a term that is well-defined within the field of stem cells.
As used herein the term “EF cells” means “embryonic fibroblast feeder”. These cells could be derived from any mammal, such as mouse or human.
By the terms “feeder cells” or “feeders” are intended to mean cells of one type that are co-cultured with cells of another type, to provide an environment in which the cells of the second type can grow. The feeder cells may optionally be from a different species as the cells they are supporting. The feeder cells may typically be inactivated when being co-cultured with other cells by irradiation or treatment with an anti-mitotic agent such as mitomycin c, to prevent them from outgrowing the cells they are supporting.
As used herein the term “QPCR” is referring to “quantitative real-time PCR”, the term “E” is referring to the PCR efficiency which can be derived as described in Example 5, the term “Ct” is referring to the cycle of threshold, terms well-known to any person skilled in the art.
As used herein the term “KRS” is referring to the relative sensitivity constant which among other things accounts for differences of fragment lengths of templates.
As used herein a “two-gene ratio” is referring to a ratio as calculated using Equation 1 or 2 herein described by the incorporation of one gene being down regulated and one gene being up regulated. The two-gene ratio or two-gene index, used interchangeably, can be calculated for one cell line at different time points or for two or more individual cell lines at the same point enabling to compare and quantify the differentiation state.
As used herein a “three-gene ratio” is referring to a ratio as calculated using Equation 1 herein described by the incorporation of at least one gene being down regulated and at least one gene being up regulated. The three-gene ratio or three-gene index, used interchangeably, can be calculated for one cell line at different time points or for two or more individual cell lines at the same point enabling to compare and quantify the differentiation state.
The following description gives instructions on suitable procedures, solutions, reagents, time periods etc. However, based on the general description and guidance herein, a person skilled in the art may vary the different elements within the scope of the invention.
The invention is further illustrated in the following examples, which are not intended to limit the invention in any way. Specific embodiments appear in the appended claims.
5 days old hIBS cell colony of five different cell lines were cut in pieces with a size of 0.1-0.3×0.1-0.3 mm, using a stem cell cutting tool (Swemed Labs International, Billdal, Sweden) and placed on mouse EF cells in 20 dishes per cell line Between 10 and 18 pieces were placed in each dish. The hBS cells were cultured in KO-DMEM media supplemented with 20% serum replacement, 1% penicillin, 1% glutamax, 1% nonessential amino acids, 0.1 mM β-mercaptoethanol and 4 ng/ml bFGF for 4-5 days (10 dishes per cell line), 14 days (5 dishes per cell line) and 21 days (5 dishes per cell line) respectively, without passaging. 50% of the media were changed every 2nd-3rd day. The hBS cell colonies were cut out mechanically (as above), transferred to 1.5 ml sterile tubes and washed in PBS. Cell pellets were stored in −80° C.
The 4-5 days old hBS cells display the morphology of tightly packed round shaped cells with a high nucleus to cytoplasm ratio, which are the typical characteristics for undifferentiated hBS cells. On the contrary, 14 and 21 days old hBS colonies displayed very different morphologies such as cystic-like structures, neuron-like structures and circle-like patterns within the colonies (see
In order to obtain spontaneously differentiated high dense hBS cell cultures, hBS cells were cultured as in Example 1 for up to 24 days without passaging. Undifferentiated and spontaneously differentiated hBS cell colonies were fixed after 4-5 days, 9-10 days, 16 days and 22-24 days, in 4% PFA for 15 minutes and subsequently permeabilized in 0.5% Triton X-100 for 5 minutes, After washing in PBS and blocking in 10% milk for 30 minutes in room temperature, the cells were incubated with primary antibodies against following antigens: SSEA-1 (1 μg/ml), SSEA-3 (1 μg/ml), SSEA-4 (1 μg/ml), TRA-1-60 (1 μg/ml), TRA-1-81 (1 μg/ml), Oct-4 (1 μg/ml), Nanog (1 μg/ml), and AFP (1 μg/ml), for 20 h in +4° C. The cells were then washed in PBS and incubated with FITC- or Cy3-conjugated secondary antibodies mixed with DAPI (0.5 μg/ml, for nuclei visualization) for 1 h in room temperature, followed by washing in PBS and mounting. The stained hBS cell colonies were visualized in an inverted fluorescence microscope. For semi-quantitative evaluation, an hBS cell colony was scored as positive if >50% of the cells in the colony were stained by the antibodies used.
Semi-quantitative marker analysis of hBS cells indicated that the expression of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and Nanog was decreased in spontaneously differentiated hBS cell colonies compared to undifferentiated cells. The expressions of these antigens never disappeared completely within the time for differentiation used in this study, though. Notably, the expression levels were still high even after 9-10 days, even though some morphology changes of the colonies were observed at that time point. It was also shown that SSEA-1 and AFP were not expressed in undifferentiated hBS cell colonies but after 16 days these antigens appear in differentiated colonies (see
Undifferentiated and spontaneously differentiated hBS cells (SA001 and SA002) maintained on EF cells were mechanically dissociated into 1×1 mm clusters using a Stem Cell Tool. The hBS cell clusters were placed in suspension cultures and allowed to form and grow as embryoid bodies (EB) (Itskovitz-Eldor et al 2000 Mol Med) in KO-DMEM media supplemented with 20% FBS, 1% PESTS 1% glutamax, 1% nonessential amino acids and 0.1 mM β-mercaptoethanol. After 2 days the number and morphology of the EB formed were evaluated. Three dimensional, homogenous EB with high cell density were scored as ++, whereas EB that attached to the culture dish and with low cell density or with irregular shape were scored as +.
A common feature for undifferentiated hBS cells is their ability to form simple and cystic EB harboring derivatives of the three embryonic gem layers when grown in suspension. We wanted to investigate if the degree of differentiation of the hBS cells was correlated to their ability to form organized EB. To this ends hBS cell clusters from undifferentiated and differentiated colonies were manually dissected and transferred to suspension cultures. After 2 days the number of EB formed and their morphology was evaluated and graded as described in above. Representative illustrations of the appearance of the EB of the various grades are shown in
Evaluation of the EB formation potential by undifferentiated and differentiated hBS cells. The cells (SA001 and SA002) were mechanically dissociated and transferred to suspension cultures. After 2 days the EB formation was evaluated as described above. The figures shown in the Table below represent the ratios between the number of ++EB formed and the total number of cell clusters originally placed in the suspension cultures. The calculated percentage values are indicated within parenthesis.
Primer design
Gene sequences were located and analysed using Ensembl (http://www.ensembl.org/). Primers were designed using the Primer3 web-based software
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and obtained primers were evaluated using Netprimer (http://www.premierbiosoft.com/netprimer/netprlaunch.html) and Beacon Designer 2.1 (Premier Biosoft). When possible, primers were designed to span intron-exon boundaries to ensure that genomic DNA was not quantified. Primers were designed for 60° C. annealing.
To ensure specificity, all primer sequences were searched in the NCBI sequence database using
BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).
To evaluate accessibility of the obtained primer sequences the resulting amplified sequence (amplicon) was analyzed using mFold
(http://www.bioinfo.rpi.edu/applications/mfold/old/dna/form1.cgi) showing all probable secondary structures at certain conditions. The amplicon melting temperature was also analyzed using the Oliqonucleotide Properties Calculator
(http://www.basic.nwu.edu/biotools/oligocalc.html).
Resulting primer sequences (depicted 5′-3′) and amplicon size were:
PCR was performed under standard conditions to generate PCR product. PCR product of 2 pooled reactions was purified using Oiagen Qiaquick PCR Purification Kit according to the manufacturer's instructions. Elution was performed in 30 μl of EB buffer.
Determination of the DNA concentration in copies/μl of the obtained PCR product was performed on the NanoDrop ND-1000 using absorbance at 260 nm. Using the amplicon size and the absorbance the concentration could be calculated. Using the purified PCR product of each PCR system, the PCR was optimized by varying the concentration of MgCl2, deoxynucleotides and primers to obtain a good PCR efficiency and as little primer-dimers as possible. All PCR reactions were performed in the Rotorgene 3000 (Cobett Research) using SYBR Green I as a reporter dye to monitor amplification during the reaction. Threshold was set above the noise and in the exponential phase of the amplification.
PCR efficiency was determined by making a serial dilution of PCR product. The Rotorgene software plots threshold cycle (the amplification round were the fluorescent signal crosses the threshold level) versus concentration and calculates the PCR efficiency, which is used in the mathematical model Eq. 1.
Melt curve analysis and gel electrophoresis was performed to ensure that the correct product had been amplified.
Extraction of total RNA from cells was performed using Qiagen RNeasy Mini Kit according to the manufacturer's instruction. During extraction the samples were DNase treated to remove any contaminating genomic DNA. DNase treatment was performed on-column using Qiagen RNase-free DNase Kit according to instructions. RNA was eluted In 30 μl RNase-free water.
The eluted RNA was quantified and checked for quality using the NanoDrop ND-1000. Absorbance spectra from 220-750 nm were measured. Quality was determined using the A260/A280 and A260/A230 ratios and concentration was determined using A260 and the NanoDrop software Concentration was determined as the average of 2 separate measurements.
Reverse transcription was performed on 1 μg of total RNA in a final volume of 20 μl using Bio-Rad iScript First Strand Synthesis Kit according to the manufacturer's instructions. The reverse transcription was run in the Rotorgene 3000 (Corbett Research).
The method described herein is not dependent on normalizing the input RNA amount and different RNA input for different samples has been used with success. Also, the reverse transcription reaction can be uniformly scaled down to 10 μl.
No RT controls were performed on each cell line by adding water instead of RT enzyme to the reaction. This controls for the presence of genomic DNA.
Each RNA sample was reverse transcribed in duplicate to evaluate the variability of the entire method.
QPCR was performed under following conditions. All genes were quantified in the same run.
OPCR reactions were run in the Rotorgene 3000 (Corbett Research). After an initial denaturation/activation step of 3 min at 95° C. followed 45 cycles of 20 s at 95° C., 20 s at 60° C. and 20 s at 72° C. Detection of fluorescent signal was performed at 72° C. in each cycle. After cycling melt curve analysis was performed by ramping the temperature from 55° C. 95° C. and monitoring fluorescence each 0.5° C. Ct values were calculated by the Rotorgene software.
Quantification of gene expression was based on the Ct (threshold cycle) value for each respective sample. When comparing several genes following mathematical model was used:
E is the PCR efficiency, Ct is the threshold cycle, and n and (m-n) are the numbers of genes that are down- and up-regulated, respectively, upon differentiation of hBS cells. The PCR efficiencies were evaluated from dilution series of purified PCR products. KRS is the relative sensitivity constant that, among other things, accounts for the differences in templates' fragment lengths. It does not affect relative comparisons of samples and was not determined.
hBS cells from five different cell lines (SA001, SA002, AS034, AS034.1 and SA121) were cultured and spontaneously differentiated as described in example 1. Primer design, optimization, RNA extraction, reverse transcription and QPCR were performed as described in the examples above and the following Ct values were obtained:
The Ct value of Oct-4, Nanog, Cripto, DNMT3B, AFP, Nestin, Desrnin OC90 and Lin28 for SA121 is the mean value (±S.D) of one PCR reaction from 2 separate RT reactions.
For all other samples it is the mean value (±S.D) of one PCR reaction in duplicate from 2 separate RT reactions.
Plotting Ct (
The genes indicated above are all suggested to be markers for undifferentiated hBS cells or their differentiated progenies. Based on the results from the PCR analysis (see Ct-values) it is obvious that some genes are better suited as reporter genes than others. A reporter gene should have the following basic criteria: a) It should be either up or down regulated upon differentiation of the hBS cells and b) it should display a similar expression profile in several (e.g. more than 2) different hBS cell lines. Furthermore, when selecting a panel of reporter genes it is of importance that the combination of genes is chosen appropriately in order to maximize the sensitivity of the assay.
In order to obtain a quantitative measure of the relative level of differentiation of the hBS cells, the mathematical model (Eq. 1) was applied. The input in the equation is the Ct-values for each individual reporter gene (Oct-4 Nanog, Cripto, and AFP) and the corresponding PCR-efficiency, and the output is an index based on the geometric averages between the RNA levels of down- and up regulated genes. By including several genes, the assay becomes very robust, while maintaining a high precision, and less sensitive to minor fluctuations in the expression levels of the individual genes.
Here we show that the combination of Cripto and AFP is clearly sufficient to discriminate between undifferentiated hBS cells and early derivatives thereof. Other combinations of potential reporter genes do not provide the same sensitivity with highest ratio in the summary table below.
Two-Gene Ratios Compared at Day 4-5 and Day 14:
The tables below show how many times higher the two-gene index is for undifferentiated material (4-5 days in culture) compared to after 14 days in culture. The last 5 genes (nestin, desmin, OC90, Lin 28, DNMT3B ) were only tested on one of the cell lines. Of the tested genes Beta-III-tubulin, nestin (both ectodermal) and desmin (mesodermal) are genes that genes that are being up-regulated according to the literature searches performed, whereas Gdf-3, OC90, Un28 and DNM3TB are genes that are being down-regulated according to the literature searches (normally expressed in undifferentiated hESCs). Although, no combination is superior in all cell lines, AFP and Cripto is the combination generating the over-all highest index ratio. The diagonal values 1.0 are obtained by inserting e.g. AFP both in the numerator and the denominator, whereby the index both at day 5 and day 14 is 1.
Three-Gene Ratios Compared at Day 4-5 and Day 14:
The tables below show how many times higher the three-gene ratios are at day 4-5 compared to day 14 for the five different cell lines by extending the two gene ratios generated as above for the combination AFP and Cripto (Shown in the last column of each table). Apart from DNMT3B which was measured only in one cell line it is shown that Oct-4 seems to give the best result compared to the other genes presented.
The calculations as presented in this example can of course be further extended to include even more reporter genes. If such reporter genes are selected arbitrarily without prior testing there is a significant risk that the sensitivity and robustness of the assay is compromised.
hBS cells were cultured as in example 1 for 7 days without passaging. Almost 300 colonies that by visual inspection appeared both morphologically undifferentiated and differentiated were cut out mechanically, using a stem cell cutting tool (Swemed Labs International, Billdal, Sweden), as whole colonies. The colonies were dissociated in 0.5 mM EDTA for 30 minutes into a single cell suspension and after centrifugation resuspended in PBS, (containing 0.1% BSA), to a final concentration of 100 000 cells/ml. The cell suspension were mixed with SSEA-4 coated magnetic Dynabeads (CELLection Pan Mouse IgG, Dynal, Norway) in a cell to bead ratio of 1:25 and incubated for 1 h at +4° C. The SSEA-4 positive cells bound to the beads were separated from the negative cells in a magnetic stand, and washed in PBS (containing 0.1% BSA). Positive and negative cell fractions were stored in −80° C. RNA extraction, reverse transcription, QPCR, and quantifications were performed as described in the examples above.
Approximately 60% of the cells were captured by the SSEA-4-antibody coated beads. The results from the QPCR analysis are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| PA 2005 00333 | Mar 2005 | DK | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP06/02159 | 3/6/2006 | WO | 00 | 11/8/2007 |
