WO2009087681A2

WO2009087681A2 - Methods for characterisation of mammalian embryonic stem cells by multiplex pcr

Info

Publication number: WO2009087681A2
Application number: PCT/IN2008/000868
Authority: WO
Inventors: Krishna Murali; Pal Rajarshi; Khanna Aparna
Original assignee: Reliance Life Sciences Pvt. Ltd.
Priority date: 2007-12-28
Filing date: 2008-12-26
Publication date: 2009-07-16
Also published as: WO2009087681A3

Abstract

The present disclosure relates to a rapid, cost effective, robust and sensitive method for routine testing of embryonic stem cells. The present disclosure in particular provides a simple inexpensive and definitive multitasked semi-quantitative multiplex RT-PCR system for human ES cell characterization.

Description

METHODS FOR CHARACTERISATION OF MAMMALIAN EMBRYONIC STEM CELLS BY MULTIPLEX PCR.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of Indian Provisional Patent Application Serial Number 2594/MUM/2007, filed on December 28, 2007, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to methods of characterization of embryonic stem cells. In particular the present disclosure is related to a multiplex RT-PCR (mxPCR) system for human ES cell characterization.

BACKGROUND OF THE INVENTION

A stem cell line comprises a population of cells that can replicate themselves for long periods of time in vitro, meaning out of the body. These cell lines are grown in incubators with specialized growth factor-containing media, at a temperature and oxygen/carbon dioxide mixture resembling that found in the mammalian body. Embryonic stem cell lines, both human and mouse, can be grown indefinitely in vitro if the correct conditions are met. Importantly, these cells continue to retain their ability to form different, specialized cell types once they are removed from the special conditions that keep them in an undifferentiated, or unspecialized, state.

Human embryonic stem cells were discovered in 1998 (Thomson, et ai, Science 282:1145— 1147, 1998). The lessons learned from working with mouse embryonic stem cells are rapidly being transferred to human embryonic stem cell systems. Pluripotent human embryonic stem (hES) cells have been shown to be derived from the inner cell mass of blastocysts and have the capacity for extensive undifferentiated proliferation in vitro (Thomson, et ah, 1998, supra; Reubinoff, et ai, Nat. Biotechnol. 18:399-404, 2000; Cowan, et ai, N. Engl. J. Med. 350:1353-1356, 2004).

In addition to their capacity of spontaneous differentiation in vitro, hES cells can be coerced to differentiate in specific pathways forming a variety of specialized cell types. However, the subtlety of how to control and manipulate hES cell differentiation to produce exclusive populations of specific cell types still remains elusive. Scientists are working hard to understand the properties of these cells and to delineate the mechanisms that regulate their differentiation into adult cell types. In addition, many researchers are using these cells to set up models to study early human development and also to provide genetic and cell-based therapies for disease.

Some of the primary experiments that still remain to be explored include: those aimed at understanding the factors required to make embryonic stem cells differentiate into the desired cell types; those to understand how to increase the number of stem cells that are accepted by the patient at the correct location in the body during disease; those to reduce host resistance to the new stem cells; and those to ensure that the new stem cells correctly integrate in the body to restore the proper function to the damaged tissue.

Besides their importance in basic research, hES cells and their differential derivatives hold tremendous promise in cell replacement therapies (Pera, et al, J. Cell Sci. 113:5-10, 2000). In addition, the hES cell technology platform is being realized to bear enormous potential in novel approaches for drug discovery and in vitro toxicity screening (McNeish, Na/. Rev. Drug Discov. 3:70-80, 2004; Davila, et ai, Toxicol. Sci. 79, 214-223, 2004). Stem cells are generally grown in culture dishes in incubators at body temperature (37°C) under high humidity. Because there are many different types of stem cells, the components of the culture solutions for each type of stem cell are different. The challenge for scientists is to grow enough stem cells in an undifferentiated state, that is without having them differentiate into more specialized cell types, and also to learn how to make the cells differentiate into specialized cells, when that becomes necessary.

Typically, hES cells are grown as small colonies on layers of skin cells in the presence of serum from the blood. The skin cells are known as "feeder cells," and together with the serum provide unknown factors that nourish and support the hES cells in their undifferentiated state. When the colonies of hES cells grow too big for their culture dishes, they are divided into smaller colonies, or single cells, and transferred into new culture dishes. The cells then continue to grow. This transfer process, known as "passaging," can theoretically be repeated indefinitely. Maintenance of hES cells in an undifferentiated state in vitro has traditionally been done on feeder layers derived from mouse embryonic fibroblasts (MEF) and human embryonic or foreskin fibroblasts (HEF/HFF) (Thomson, et al, 1998, supra; Reubinoff, et al, 2000, supra; Richards, et al, Stem Cells 21:546 -556, 2003; Amit, et al, Biol. Reprod. 68:2150-2156, 2003; Hovatta, et al, Hum. Reprod. 18:1404-1409, 2003). In addition, feeder-free conditions for maintaining hES cells have also been reported (Xu, et al, Nat. Biotechnol. 19:971-974, 2001; Amit, et al, Biol. Reprod. 70:837-845, 2004). Presently, stem cell scientists are closely considering establishment of a complete xeno-free defined culture system for growing hES cells. Furthermore, efficient propagation of hES cells depends critically on the time of passaging the cells, which is commonly 4-7 days. However, despite standardized culture conditions, spontaneous differentiation in hES cells in culture may occur very frequently rendering their maintenance in undifferentiated state challenging.

Differentiating hES cells can be identified based on changes in morphology of cells accompanied with down regulation of stem cell specific markers and concomitant up regulation of markers associated with differentiated phenotypes (Pera, et al, 2000, supra; Draper and Fox, Arch. Med. Res. 34:558-564, 2003). Further, various labor intensive, time consuming, expensive tests can be performed to demonstrate pluripotency of hES cells in vitro (Carpenter, et al., Cloning Stem Cells 5:79-88, 2003; Bhattacharya, et al, Blood 103:2956-2964, 2004; Brimble, et al, Stem Cells Dev. 13:585-597, 2004; Josephson, et al, BMC Biology 4:28-40, 2006).

Reports from independent groups have suggested that the human ES cell lines studied to date are somewhat similar in the expression of certain molecular markers implicating "sternness" (Hoffman and Carpenter, Na/. Biotechnol. 23:699-708, 2005). Although not all markers have been tested in all lines, expression of a set of markers associated with pluripotency and differentiation potential is accepted as a standard for most hES cell lines. However, it is obvious that there are indeed some differences in the gene expression profiles between individual hES cell lines. This is not surprising since all hES cell lines are derived from different embryos, each representing a unique genetic history. In addition, differences in culture conditions adopted by various laboratories, which prompts certain genomic and epigenomic changes within the cell line, may also lead to great difficulty in comparing data from one laboratory with another and sometimes even with different passages of the same line.

It is clear that routine characterization of hES cell lines is essential to avoid compromising the validity of results. One of the most frequent modes of characterization is semi quantitative RT-PCR, which is used for those genes whose expression is involved in maintenance of the undifferentiated state. Meanwhile, novel stage-specific genes that distinguish between hES cells and embryoid bodies (EBs) have been identified (Maitra, et al, Nat. Genet. 37:1099-1103, 2005; Cai, et al, Stem Cells 24:516-530, 2006; Bhattacharya, et al, BMC Dev. Biol. 5:22-37, 2005). Thus, the list of genes that can be considered as common molecular markers for undifferentiated hES cells is relatively small and well documented.

On the other hand, derivatives of hES cells can be perceived by a number of genes that are expressed exclusively by differentiated cell types. Furthermore, since differentiated cells are often diffused within or towards the edge of colonies and differentiation is subtle, it is difficult to detect by inspection of morphological characteristics, and even by immunohistochemistry. Earlier characterization schemes of hES cells included RT-PCR, immunochemistry, karyotype, HLA and STR analysis, DNA fingerprinting, telomerase assay, teratoma formation in SCID mice, real-time PCR, focused cDNA microarray- miRNA- and mitochondrial DNA analysis (Mandal, et al., Differentiation 74:81-90, 2006; Pal, et al, Regen. Med. 2:179-192, 2007).

Although several molecular methods like real-time PCR (QPCR), whole genome microarray and microRNA analysis, EST scan, SAGE, MPSS have proved to be increasingly sensitive and efficient for monitoring hES differentiation, most of these high-throughput tests have a limited use due to high cost and extended turn-around time (as a service), coupled with an overwhelming demand for highly-specialized technical expertise (in-house). Thus, there is a general lack of rapid, cost-effective, robust, yet sensitive methods for routine testing of ES cells.

As it is well accepted that culturing of hES cells calls for caution and more so in order to restrict them from spontaneous differentiation during long term maintenance, the importance of subjecting hESC to routine characterization has also been emphasized (as reviewed by Loring and Rao, Stem Cells 24:145-150, 2006). This is because these cells in culture tend to accumulate certain changes in their genomic and epigenomic signature (Maitra, et al., 2005, supra; Bibikova, et al., Genome Res. 16:1075-1083, 2006; Liu, et al., 2007). Likewise, subtle changes in the gene expression profile of hESC may alter the overall characteristics of a cell line by impacting its developmental competency in vitro, thereby rendering it unsuitable for cell replacement therapy. Hence, it is imperative to consider routine characterization of hESC with an interval of every five passages, at least at a preliminary level.

There is a need for an explicit and unambiguous data set from gene expression analysis of hES cells at different stages, irrespective of the origin and culture conditions for their successful propagation and expansion in the laboratory. Further the gene expression analysis also provides the differentiation competency of a particular cell line into ecto-, meso- or endoderm, depending on the expression of key lineage specific markers. In view of a large number of laboratories venturing into ES cell research and the emerging potential of these ES cells in regenerative medicine and drug discovery, a reliable and robust, yet simple and rapid screening method for routine characterization of hES cells is clearly needed.

OBJECT OF THE INVENTION

It is an object of the present invention to provide an assay method for characterization of all mammalian pluripotent cells and in particular embryonic stem cell lines.

It is an object of the present invention to provide a method for evaluating differentiating ES cells.

It is an object of the present invention to provide a semi-quantitative method of evaluation of

ES cells.

It is an object of the present invention to provide a single reaction multiplex RT-PCR technique for semi-quantitative evaluation of differentiating cells.

It is an object of the present invention to provide a set of at least fourteen markers for comparing the mRNA levels in a single PCR technique.

It is an object of the present invention to avoid false negative and false positive results by inclusion of appropriate internal controls.

It is an object of the present invention to provide a method that can be performed in less time with a smaller amount of reagents.

It is an object of the present invention to provide a method for qualitative and quantitative assessment of the cells.

It is an object of the present invention to provide a simple, rapid, cost effect, robust and yet sensitive method for routine testing of all pluripotent mammalian cells.

SUMMARY OF THE INVENTION

The present disclosure provides a reliable and robust, yet simple and rapid screening method for routine characterization of hES cells. In addition, the present disclosure provides a simple and effective, semi-quantitative RT-PCR analysis of a candidate set of gene markers associated with pluripotency as well as differentiation. In this regard, the present disclosure details the development of multiplex RT-PCR (mxPCR) strategies for rapid and cost effective screening of mammalian pluripotent stem cells, in particular hES cells, that may be significant in defining the state of cells. Thus the present disclosure provides a method of characterization of all mammalian pluripotent cells, and in particular human ES cells, and provides assay methods that are rapid, cost effective and commercially viable. The present disclosure also provides a single-reaction multiplex RT-PCR, for semiquantitative evaluation of differentiating hES cells. In certain embodiments, the methods involve the comparison of relative mRNA levels of a set of about fourteen markers in hES cells and/or human embryoid body (hEB) samples so it is possible to discriminate between undifferentiated hES cells and their early derivatives on the basis of up- and/or down- regulation of a set of biomarkers implicating diverse functions. In other embodiments, , the methods involve the comparison of relative mRNA levels of a set of at least about five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty markers in hES cells and/or human embryoid body (hEB) samples. The present disclosure also provides a simple, rapid, cost effect, robust and yet sensitive method for routine testing of all pluripotent mammalian cells. The present disclosure provides the design and development of multiplex PCR for characterization of the embryonic stem cells, for example hES cells.

The present disclosure provides a simple, rapid, robust, inexpensive and definitive multi- marker semi-quantitative method for characterization of the ES cells by using a single- reaction mxPCR method. This single reaction mxPCR method is flexible, and by selecting appropriate reporter genes, can be designed for important applications including routine characterization of different hES cell lines maintained in various laboratories, and in pharmacology and cytotoxicity screening.

In one aspect the present disclosure provides a single multiplex RT-PCR for evaluation of the differentiating cells. In an attempt to understand the changes in gene expression pattern of hES cells preceding differentiation, the present disclosure provides a simple, rapid, robust, inexpensive and definitive multi-marker semi-quantitative multiplex RT-PCR (mxPCR) system.

In one aspect the present disclosure provides a multiplex PCR designed with careful consideration for the regions to be amplified, relative size of the fragments, and the dynamics of the primers. In other aspects the present disclosure provides multiplex PCT methods in which internal controls are employed to ensure the integrity of the cDNA templates. In one aspect the present disclosure provides a method that can be performed in less time with a minimum amount of reagents. In one particular aspect the frequent usage of a thermal cycler was reduced. In another aspect the present disclosure provides an assay method that gives both a qualitative and a quantitative assessment of hES cells.

In another aspect the present disclosure relates to the comparison of the mRNA levels using, for examples, at least fourteen markers to discriminate between undifferentiated hES cells and their early derivatives. In order to maximize the sensitivity of the test, methods of the present disclosure measure the expression of genes that are up- and down-regulated before and after differentiation of hES cells. Out of genes assayed (e.g., eight undifferentiated markers in two sets and six differentiated markers in two sets) it was observed that the method was clearly sufficient to determine the relative differentiation state of hES cells in culture.

In still another aspect the present disclosure provides a combination of RT-PCR and related hES cell-based technologies to provide a tool for routine analysis in a cost effective manner.

The present disclosure evaluates the method using two independent hES cell lines

(ReliCell®hESl and BGOl), and also in a human embryonic carcinoma line (NTERA-2), thereby demonstrating that the method is very simple, rapid, robust and generally applicable for all cell lines tested. The combination of RT-PCR and related hES cell-based technologies provides a useful tool for routine analysis of hES cells in a cost-effective manner.

In one aspect the present disclosure provides a mxPCR assay as a tool in determining spontaneous differentiation during conventional maintenance of hES cells. In one particular aspect the present disclosure permits distinction among undifferentiated and committed cells, through differential gene regulation.

In one aspect the present disclosure provides a method that is able to monitor the state and the purity of the hES cell population in a fast, accurate and sensitive way, compared to other conventional techniques.

In one aspect the present disclosure provides an assay method that is relatively affordable and can be made available in countries with limited resources, unlike other advanced molecular methods like Q-PCR-, microarray-, and microRNA-analysis, SAGE and MPSS.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1: Morphological and immunological characterization of hESC and EBs generated from ReliCell®hESl. Phase contrast images of (A) tightly packed 5 day old undifferentiated ES colonies from ReliCell®hESl at passage 37 maintained on MEF and; (B) day 15 EBs in suspension culture growing in ultra-low adherent bacteriological dishes. Undifferentiated hESC plated on 1% matrigel coated 2 well-chambered slides stained against (C) Oct-4 and (D) SSEA-4 antibodies showed uniform positive green fluorescence all over the ES colony; day 15 EBs immunostained against (F) Nestin and (G) GATA-4, a neuroectoderm and mesendoderm marker respectively; cellular distribution of GATA-4 protein was confined to the border of the EBs. DAPI was used as a counter stain (E, G). Scale bars range from 50-100 μm.

Figure 2: Development of mxPCR system with undifferentiated cells of ReliCell^®hESl. (A) Lane 1 : mxPCR with set 1 comprising of a subset of 4 pluripotent undifferentiated ES markers namely TDGFl, Sox2, Rexl, and Thyl, lane 2 to 6: uniplex PCR using the same set of genes, lane 7: (-) template control; (B) lane 1 : mxPCR with set 2 consisting of another 4 ESC markers namely Cx-45, Dppa5, Nanog, and Cripto, lane 2 to 6: uniplex PCR using the same set of genes, lane 7: (-) template control; (C) lane 1: mxPCR with set 3 comprising of a combination of early differentiation markers including Kit- 18, c-actin, and HNF-3β, lane 2 to 5: uniplex PCR with the same set of primers, lane 6: (-) template control; (D) lane 1: mxPCR with late stage differentiation markers including NFH, Msxl, and Albumin grouped as set 4; lane 2 to 5: uniplex PCR with the same set of primers, lane 6: (-) template control. (C & D) Noticeably, the expression of differentiation (ecto-, meso- and endoderm) markers in day 5 hESC is distinctly down-regulated. For each mxPCR set developed GAPDH was used as an internal control and M represents 100 bp molecular marker.

Figure 3: Simultaneous detection of early differentiation markers in ReIiCeIl^hESl- derived EBs by mxPCR assay. (A) Lane 1: mxPCR with set 1; lane 2 to 6: uniplex PCR using the same set of genes, lane 7: (-) template control; (B) lane 1 : mxPCR with set 2; lane 2 to 6: uniplex PCR using the same set of genes, lane 7: (-) template control; (C) lane 1: mxPCR with set 3, lane 2 to 5: uniplex PCR with the same set of primers, lane 6: (-) template control; (D) lane 1: mxPCR with set 4; lane 2 to 5: uniplex PCR with the same set of primers, lane 6: (-) template control. (C and D) Expression of the early differentiation genes (Krt-18, Msxl, HNF-3β) and markers for mature tissues (NFH, c-actin, Albumin) are significantly enhanced compared to pluripotent markers of set 1 and 2 in day 15 EBs (A and B). For each mxPCR set GAPDH was used as an internal control, and M represents 100 bp molecular marker.

Figure 4: Establishment of mxPCR as a sensitive method for defining the state of hESC by testing a commercially available control cell line, BGOl. (A) and (B) represents 2% agarose gel images from mxPCR analysis of RNA isolated from undifferentiated cells of BGOl at passage 42 using set 1 & 2, constituting of 8 pluripotent ESC markers. (C) and (D) represent mxPCR results using RNA from day 15 EBs of the same cell line with set 3 and 4, which comprises 6 lineage and tissue specific markers. GAPDH was used as a housekeeping gene. The gene expression pattern in BGOl is in concordance with the profile obtained in case of ReliCell®hESl, which confirms the sensitivity and reproducibility of the developed test system and highlights the utility of these set of biomarkers as likely initial candidate genes for routine characterization of hES cells in any lab.

Figure 5: Validation of mxPCR strategy and trouble shooting. (A) mxPCR analysis with set 1-4 using RNA from HEF failed to detect the expression of any genes except GAPDH demonstrating that the individual primer sequences used in this study are specific to the target gene and are not expressed indiscriminately. (B) mxPCR analysis of day 21 differentiated EBs post-induction to cardiomyogenic lineage by BMP-2 treatment, shows conspicuous expression of mesodermal markers (Msxl and c-actin) and few early differentiation genes like Kit- 18 and NFH. (C) Elimination of non-specific products for set 1 in absence of cDNA template by reducing the primer concentration from 40 nmol to 30 nmol for Sox2, 30 nmol to 20 nmol for Rexl, 20 nmol to 10 nmol for Thyl (lane 3 and 4) accompanied with a I⁰C rise in the Tm (55-56⁰C); lane 5 and 6 shows that at 57⁰C the non-specific band reappeared. (D) At 3 different Tm (53.5, 54.5 and 55.5°C) expression of HNF-3β was masked due to over expression of Kit- 18 (lane 1-3), which was resolved by decreasing the primer concentration of Kit- 18 from 8 nmol to 4 nmol at 54.5 or 55⁰C (lane 5 and 6); in lane 4 expression of HNF- 3β was further confirmed by uniplex PCR. These data indicate the importance of adopting stringent conditions in mxPCR approach.

Figure 6: Validation of the technique using the human ES cell lines RelicellhES 2, 3 and 4. The same results were found as those for Relicell®hESl and BGOl.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

The term "pluripotent stem cells," as used herein, refers to cells that have high self-renewal capacity and possess differentiation potential, both in vitro and in vivo. A pluripotent cell can be self-renewing and can remain dormant or quiescent within the tissue or organ.

The term "mammalian," as used herein, refers to various warm blooded vertebrates, animals of class mammalia, including humans, characterized by a covering of hair on the skin and in the females, milk producing mammary glands for nourishing the young.

The term "multiplex PCR," as used herein, refers to a PCR reaction where more than one primer set is included in the reaction pool allowing two or more different DNA targets to be amplified by PCR in a single reaction tube. The present disclosure provides the design and development of multiplex PCR for characterization of mammalian pluripotent stem cells, in particular human embryonic stem cells. The design and development of multiplex PCR is based on combining sets of primers for which reaction conditions have been determined separately. The present disclosure provides multiplex PCR that is designed on aspects including, but not limited to, the region of amplification, the relative size of the fragment, and the dynamics of the primers, as shown in Table 1. The methodology set forth below is exemplary of the present disclosure, arid can be varied using the knowledge of one of skill in the art for the characterization of mammalian pluripotent stem cells without undue experimentation.

The steps involved in developing the multiplex PCR technique comprise the following: (1) develop PCR conditions separately for each primer set; (2) add primer sets sequentially, altering conditions as necessary, reduce nonspecific amplification; and (3) adjust reaction components and cycling conditions for multiplex amplification. The development of PCR conditions was done separately for each primer set. The primer sets were then added sequentially wherein the conditions were altered as required to reduce the non-specific amplification by hot start, ionic detergents, short extension times, hottest annealing, reselecting primer sequence, varying relative concentrations of primer sets for equal amplification, or changing buffer systems. The reaction conditions and polymerase requirements may increase, and ideal extension times may be longer. Typical Multiplex PCR was performed in a 0.2 ml eppendorf tube consisting of 25 μl of 2x PCR Master Mix (AB gene contains 1.25 units of thermoprime plus DNA polymerase, 75 mM Tris-HCl (pH 8.8 at 25°C), 20 mM (NH₄)₂ SO₄, 1.5 mM MgCl₂, 0.01% (v/v) Tween® 20 and 0.2 mM each of dATP, dCTP, dGTP, and dTTP).

Internal controls: Although cDNAs were initially screened for GAPDH expression as a quality control, it was important to include an internal quality control (house keeping gene) to ensure the integrity of the cDNA templates. Therefore, the present disclosure included GAPDH in all the sets of mxPCR system.

Template quantity and quality: 2 μl cDNA was added in each set of reactions, which were prepared using 1 μg RNA isolated from human ES cells. The concentration of RNA was estimated on a UV spectrophotometer at 260 nm. Hence, 100 ng of RNA was used for each reaction.

Less expense of time and reagents: In uniplex PCR, 12.5 μl of 2x PCR master mix and 2 μl of cDNA was used in 25 μl reaction volume per interested gene of amplification. However, the present disclosure uses a total reaction volume of 50 μl containing 25μl of 2x PCR master mix and 2 μl of cDNA for amplification of five genes in the first two sets and four genes in the remaining sets. Consumption of other reagents, such as agarose, 5Ox TAE buffer, ethidium bromide, and 100 bp DNA ladder, was relatively low. Further, frequent usage of the thermal cycler was reduced.

Using 2 μl of cDNA, and suitable primers in appropriate concentrations (Table 1), the final volume was made up to 50 μl with sterilized water. Amplification was performed up to 35 cycles involving initial denaturation at 94⁰C for 2 minutes, denaturation at 94°C for 45 seconds, annealing at a pre-determined temperature (Table 1) for 45 seconds, extension at 72⁰C for 1 minute, termination at 72°C for 10 minutes, and soaking at 4°C. The above method is more advantageous in that it is a cost-effective, robust, and sensitive method for routine testing of ES cells. This method can also be applicable for measuring the expression of genes up- and down-regulated to determine spontaneous differentiation of ES cells. Qualitative assessment of ESC: The hES cells maintained on MEF were fixed, and subsequently the cells were stained using antibodies directed against Oct-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Positive staining with these markers suggests that the hES cells used maintain their ideal pluripotent characteristics.

Quantitative assessment of ESC: Around 60-70 hES colonies, each colony consisting of around 10,000 to 20,000 hES cells, were collected and used for RNA isolation. The cells were examined for viability by trypan blue exclusion method.

The present disclosure provides a mxPCR system that allows for simultaneous and specific detection of a candidate set of genes responsible for undifferentiated and differentiated hESC in separate sets of 1-4. These genes in isolation could be clearly detected in the cells (Figures 2, 3, and 4). The present disclosure focuses on a multiplex system in which each primer pair targets a single gene/locus, unlike RAPD (Williams, et al, Nucleic Acids Res. 18:6531-6535, 1990) or alumorph (Zietkievcz, et al., Proc. Natl. Acad Sci. USA 89:8448-8451, 1992) PCR reactions.

The mxPCR methods of the present disclosure have several advantages over normal PCR on a practical standpoint. The most common problem in PCR includes false negatives due to reaction failure or false positives due to contamination. False negatives are often revealed in multiplex amplification because each amplicon provides an internal control for the other amplified fragments.

The quality of the template may be determined more effectively in multiplex than in single locus PCR. The present disclosure uses the internal standards of mxPCR to assess the amount of a particular template in a sample. The majority of multiplex quantitation assays compare the signal intensity of a reference sequence to the signal from another sequence in the same reaction, either directly or by extrapolating the result to standard curves (Ferre, PCR Methods Appl. 2: 1 -9, 1992).

Importantly, the expense of reagents and preparation time is much less in mxPCR than in conventional systems where several tubes of uniplex PCRs are used. The present disclosure provides data of 15 different biomarkers (Table 1) including GAPDH as housekeeping gene, in 4 separate sets categorized on the basis of their function. Thus a multiplex reaction is ideal for conserving expensive polymerase and templates, which are in poor supply. For maximum efficiency of preparation time, the reactions can be prepared in bulk, randomly tested for quality, and stored frozen without enzyme or template until use.

Another, important aspect of this approach is the design and development of multiplex PCR. The mxPCR system of the present disclosure is relatively simple as two or more sets of primers were combined. The present disclosure provides a method with careful consideration for the specific regions to be amplified, the relative sizes of the fragments, the dynamics of the primers, and the optimization of PCR technique to accommodate multiple fragments. The present disclosure provides PCR conditions separately for each primer set (Table 1). The present disclosure has eliminated an important ESC-related transcription factor, Oct-4, from the assay method, which essentially camouflaged the expression of other gene markers such as Rexl, Sox2, Dppa5, and Nanog (Fig. 5A, B). This is due to the presence of a redundant base (R) in the forward primer sequence of Oct-4, evoking competitive inhibition in binding of other primers to the template encoding similar bases upstream or downstream. The present disclosure has also confirmed the expression of Oct-4 by quantitative real time PCR analysis (Pal, et al, 2007, supra) and concluded that the problem is application specific and hence has no effect on the characteristic of the hES cell line. In set 1, surprisingly, expression of Sox2, Rexl and Thyl was observed in minus template controls, which was eventually overcome by a 1°C rise in annealing temperature and a decrease in the primer concentrations by 10 nmol. Similarly the inclusion of NFH, an early stage neural marker, while developing set 3, was effectively resolved with the same approach. Further, in the same set 3, it was observed that Krt-18 masked the expression of HNF-3β. HNF-3β, a marker for definitive endoderm, is barely expressed in day 15 EBs in the presence of Krt-18, an epithelial cell marker. This problem was circumvented by cutting down the primer concentration of Krt-18 from 10 nmol to 4 nmol. Interestingly, the inventors of the present disclosure noticed that during cDNA synthesis, just before addition of the reverse transcriptase enzyme, incubating the RNA mix for 2-5 minutes at 42°C ultimately produces better quality cDNA. This may be due to the time provided for RNA stabilization, which subsequently improved the substrate-enzyme reaction kinetics. Furthermore, it was observed that the most appropriate amplification times in mxPCR for the majority of the primer sets was between 30-35 cycles, which is also in concurrence with the hypothesis that the probability of non-specific products is aggravated with increase in number of amplification cycles.

The mxPCR assay of the present disclosure is a tool in determining spontaneous differentiation during conventional maintenance of hES cells, as it permits distinction among undifferentiated and committed cells, through differential gene regulation. This simple strategy is instrumental in monitoring the state and purity of an hESC population. It is fast, accurate, and sensitive, and unlike other more advanced molecular methods like Q-PCR-, microarray-, and microRNA-analysis, SAGE and MPSS, is relatively affordable in countries with limited economic resources.

The method of the present disclosure can be used to identify Duchenne/Becker muscular dystrophy (DMD/BMD) regions of deletion, and the steroid sulfatase gene detects, which are frequently whole-gene deletions, mutations, and small deletions. The method of the present disclosure may also be used for repetitive DNA polymorphisms for mapping, disease linkage, gender determination, and DNA typing/identification. The multiplex PCR reactions also have applications in paternity testing and forensic identification. Multiplexed polymorphic repeats determine whether family members have inherited an identical chromosome to the proband. Further, the present disclosure provides methods for detection of embryos of families with X-linked diseases, which can be sexed by co-amplifying a Y-specific repetitive DNA locus with a gene sequence on both X and Y-chromosomes.

The methods of the present disclosure may also be used to distinguish species of Legionella, Mycobacterium, Salmonella, Escherichia coli, Shigella, and major groups of Chlamydia or associated bacteria. An assay for Mycobacterium leprae co-amplifies human and pathogen DNA. Multiplex assays differentiate forms of the insecticidal protein crystal producing Bacillus thuringiensis, Shiga-like toxin-producing E. coli, and yeast. Viral DNA can also be amplified by multiplex PCR to screen tissue samples, or to examine associations of infection with disease (Edwards and Gibbs, PCR Methods Appl. 3:S65-S75, 1994). The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE l: CELL LINE PREPARATION

1) Maintenance and expansion of hES cell lines: Mechanical passaging of the undifferentiated hES cell colonies of ReliCell®hESl and BGOl was carried out by cutting the colonies into small clumps of about 200-250 cells using the sharp edge of a flame-pulled pasteur pipette. Caution was adopted to select only undifferentiated portions of the hES colonies in case of spontaneous differentiation during sub-culturing. The cultures were grown at 37⁰C and 5% CO₂ in air (Forma Scientific, USA). The NTERA-2 cell line was cultivated in normal DMEM (Gibco) with 10% FBS (Gibco), and for HEF normal fibroblast medium was used without LIF. Media for all cultures were replaced every alternate day.

2) Generation of embryoid bodies (EBs) in suspension culture: The hES colonies were again manually cut into small clumps of approximately 100-150 cells. These small pieces of hES colonies were transferred onto low-adherence bacteriological plates (Nunc) in media consisting of 80% DMEM/F-12 (Gibco), 15% ES tested FBS (Hyclone, USA), 5% Serum replacement (Gibco), 1% nonessential amino acid solution (Gibco), ImM glutamine (Gibco), and 0.1% beta-mercaptoethanol (Sigma). Undifferentiated hES cells spontaneously form embryoid bodies (EB) starting from day 2 in suspension, implicating the onset of differentiation. Media was replaced every alternate day until the EBs had grown in size and maturity for 15 days.

EXAMPLE 2: MULTIPLEX PCR

1) Total RNA isolation and cDNA synthesis: Test samples included hES cell lines ReliCell®hESl and BGOl. Cells were collected and total RNA was isolated by RNeasy spin column method (Qiagen) according to the manufacturer's protocol. After estimation on a spectrophotometer, 1 μg of RNA treated with RNase-OUT ribonuclease inhibitor (Invitrogen) was used for cDNA synthesis. In general, 40-50 medium-sized hES colonies and ~10⁶ human embryonic carcinoma (hEC) cells yielded approximately 3-4 μg of total RNA. Reverse- transcription using Superscript reverse transcriptase-II (Invitrogen) and OligodT (Invitrogen), to prime the reaction was carried out in a 20 μl of reaction mix.

2) PCR primers and amplification: All of the gene markers have been tested and the results reported (Mandal, et al, 2006, supra). Hence, based on the earlier experience of working with various undifferentiated or pluripotent and lineage specific markers in a semiquantitative set up (Mandal, et al, 2006, supra), PCR primers were selected to distinguish between cDNA and genomic DNA. 1 μl of cDNA was amplified by polymerase chain reaction (PCR) using Abgene 2X PCR master mix (Abgene, UK) and appropriate primers (Table 1). For all genes, amplification was performed for 35 cycles, consisting of an initial denaturation at 94°C for 1 minute, then 94°C for 30 seconds, annealing temperature of the respective gene primer (Tm) for 45 seconds, 72°C for 1 minute, and was terminated by final extension at 72°C for 5 minutes.

Table-1: Represents the list of genes along with primers sequences, annealing conditions, concentration of the primers, and region of amplification in mxPCR.

Set l

Set 2

Set -4

3) Multiplex PCR: All the selected genes were tested for amplification specificity and robustness at annealing temperatures ranging from 52°C - 6O⁰C for optimization of multiplex PCR. It was important to decrease the annealing temperature, prolong the extension time and optimize the primer concentration to eliminate cross reactions between primers with sequence homologies. Multiplex PCR was performed in a 0.2 ml eppendorf tube consisting of 25 μl of 2x PCR Master Mix (AB gene contains 1.25 units of thermoprime plus DNA polymerase, 75 mM Tris-HCl (pH 8.8 at 25⁰C), 20 mM (NH₄J₂SO₄, 1.5 MgCl₂, 0.01% (v/v) Tween® 20, and 0.2 mM each of dATP, dCTP, dGTP, dTTP). Using 2 μl of cDNA, and suitable primers in appropriate concentrations (Table 1), the final volume was brought up to 50 μl with sterilized water. Amplification was performed up to 35 cycles involving initial denaturation at 94⁰C for 2 minutes, denaturation at 94°C for 45 seconds, annealing at pre-determined temperature (Table 1) for 45 seconds, extension at 72⁰C for 1 minute, termination at 72°C for 10 minutes, and soaking at 4°C.

4) Detection of amplified Multiplex PCR product: Products were analyzed on 2% IX TAE (5OX TAE: 242 g of Trizma base, 57.1 ml of glacial acetic acid, 37.2 g of Na-EDTA) agarose gel stained with ethidium bromide (Sigma). The electrophoresis was carried out in a horizontal gel tank at 70 V for 45 minutes or until desired resolution was obtained. Gels were viewed by UV transilluminator and photographed. lOObp ladder (Invitrogen) was used as molecular weight markers in all gels.

EXAMPLE 3: RESULTS

1) Culture and differentiation of hES cells: The hES cell lines maintained on MEF and used in this disclosure have been extensively characterized previously and they express cell surface antigens and relevant transcriptional markers of hESC as well as exhibiting in vivo and in vitro pluripotency (Mandal, et al, 2006, supra; Pal, et al, 2007, supra; Brimble, et ai, 2004, supra). 5 days old hESC colonies displayed the morphology characteristic for undifferentiated hESC (i.e., large compact multicellular colonies of cells with a high nucleus- to-cytoplasm ratio and shiny borders). At this time point, the hESC cultures were passaged by mechanical dissociation. It was observed that undifferentiated hES cell colonies efficiently formed simple and cystic EBs when placed in suspension cultures up to 15 days (Fig. IB). In the presence of BMP-2 the EBs were allowed to further differentiate into cardiomyocyte-like cells for about 14-21 days as described (Pal and Khanna, Differentiation 75:112-122, 2007). The undifferentiated as well as the differentiated hES cells were collected at different points to perform the mxPCR analysis. The expression of markers indicative of ecto-, meso-, and endodermal derivatives has previously been demonstrated in these cells (Mandal, et al., 2006, supra). Taken together, these data indicate that the hES cells remain pluripotent at least up to 5 days after passage, while the cultures at day 10 and 14 consist of heterogeneous populations of undifferentiated and differentiating cells.

2) Expression of stage specific markers in hESC: The expression of a set of frequently used hESC markers were examined at the protein level by immunocytochemistry and at the mRNA level by RT-PCR. The hES cells on MEF at passage 36 and EBs were fixed, and subsequently the cells were stained using antibodies directed against Oct-4, SSEA-4, and Nestin. Representative staining patterns obtained are shown in Figs. 2C-2F. The results demonstrated that Oct-4 and SSEA-4 were expressed by the majority of the cells in the undifferentiated hES cell colonies at 4-5 days, and Nestin was expressed in 15 day old EBs. During differentiation, the expression of the ESC markers decreased. Further, RT-PCR with undifferentiated colonies from ReliCell®hESl and BGOl celMines at passage numbers 37 and 42 showed consistently that the expression of pluripotency markers, including Nanog, Rexl, Sox2, and TDGFl, was equal in both lines, while Dppa5 (developmental pluripotency associated gene 5) was lower in ReliCell®hESl than in BGOl (Fig. 2A, B and Fig. 4A, B). Conversely, no major differences were found in the expression pattern of lineage specific markers like Krt-18, NFH, Msxl, c-actin, HNF-3β, and albumin in day 15 EBs from either of the cell lines (Fig. 3B, C and Fig. 4B, C). As expected, the expression of pluripotent markers in EBs and differentiation markers in ES cells was very low or almost undetectable (Fig. 2C, D and Fig. 3A, B). Taken together, these data indicate that the hES cells remain pluripotent at least up to 5-6 days after passage, and that both cell lines are capable of differentiating into derivatives all three germ layers (ecto-, meso- and endoderm).

3) Multiplex PCR system and its detection sensitivity: Several combinations of primers were tried for simultaneous amplification of target sequences. After repeated attempts the present disclosure successfully developed a mxPCR system involving four sets of markers (Table 1) using minimal volume of sample. Among these, two sets contain pluripotent ESC markers and the rest are with differentiated markers corresponding to ecto-, meso-, and endoderm lineages. The results (Fig.2 and Fig. 3A-D) show that all four sets were sufficiently amplified under the PCR conditions described herein. In the first two sets, five different markers in each set, and in the later two sets, four different markers in each set could be amplified in a single mxPCR reaction. Control gene markers were run in parallel with mxPCR to achieve proper quantification. Although cDNAs have been initially screened for GAPDH expression as a quality control, it was important to include an internal control (housekeeping gene) to ensure the integrity of the cDNA templates. Therefore GAPDH primers were included in all sets of the mxPCR system. However, GAPDH and other primers have different amplification kinetics and these differences were circumvented by optimizing the primer concentrations (Table 1).

The detection sensitivity of the mxPCR system was also determined. The same set of experiments were repeated on the control cell line BGOl (Fig. 4A-D) and the detection sensitivity was in concurrence to the results with ReIiCeIl⁽DhESl. This mxPCR system indicates simultaneous amplification of more than one pluripotent or differentiated gene marker in a single reaction tube without any cross-reaction between primers. Table 2 describes a comparative expression analysis of the biomarkers by uniplex and multiplex PCR, which suggests that all the genes are present uniformly, but that the extent of their expression may differ in some cases like Nanog, Sox2, Cx-43, NFH, Msxl, HNF-3β, and albumin. Hence, this system could be a suitable option for rapid screening of hES cells. The validation of the technique was done using the human ES cell lines RelicellhES 2, -3 and -4 (Figure 6). The same set of results was found as those for Relicell®hESl and BGOl . Table 2: Comparison of relative levels of gene expression in hES cells by uniplex and multiplex PCR.

Further, the mxPCR system was validated by clearly demonstrating differential gene expression pattern in day 14 EBs post-treatment with penicillin (1000 μg/ml) and 5-Fluro-uracil (1 μg/ml) (Fig. 3). This finding implicates that the test developed is sufficiently sensitive to detect diminutive changes in transcript levels of the proposed set of genes.

4) Validation of the mxPCR strategy: In order to validate the mxPCR screening method, human embryonic fibroblasts (HEF) were subjected to mxPCR for all 4 sets developed. None of the markers were expressed in the HEF except GAPDH, which is a housekeeping gene (Fig. 5A). Additionally, mxPCR was performed with hES cells (passage 35) induced to cardiac differentiation at day 21 (Pal and Khanna, 2007, supra). In set 3 and set 4, an up- regulation in expression of mesodermal genes was detected, including Msxl and c-actin, over markers responsible for other lineages such as Kit- 18 and NFH (ectoderm), and HNF-3β and albumin (endoderm) (Fig. 5B). This was accompanied with a remarkable fall in the levels of pluripotent markers in set 1 and set 2. This finding implicates that the test developed is sufficiently sensitive to detect diminutive changes in transcript levels of the proposed set of genes even during differentiation to a particular phenotype.

Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, be within the scope of the invention.

Claims

1. A process for analysis of mammalian pluripotent stem cells comprising multiplex RT-PCR (mxPCR) analysis.

2. A process for the mxPCR analysis of mammalian pluripotent stem cells comprising analyzing the expression of a plurality of genes associated with pluripotency and differentiation.

3. The process according to claim 1 and 2, wherein the mammalian pluripotent stem cells are human embryonic stem cells.

4. The process according to any of the preceding claims, comprising: a) mixing cDNA isolated from the mammalian pluripotent stem cells and at least one set of primers from Table 1; b) amplifying the mixture using PCR; and c) detecting the amplified product.

5. The process according to claim 4, wherein the cDNA was synthesized from RNA isolated from the mammalian pluripotent stem cells.

6. The process according to claim 4, wherein the concentration of primers is selected to eliminate cross reaction between primers and sequence homologies.

7. The process according to claim 4, wherein the PCR mix is a master mix of AB gene comprising 1.25 units of thermoprime plus DNA polymerase, 75 mM Tris-HCl, ammonium sulfate, magnesium chloride, Tween 20, dATP, dCTP, dGTP, and dTTP.

8. The process according to claim 4, wherein the mixture is amplified for up to 35 cycles involving initial denaturation at 94⁰C for 2 minutes, denaturation at 94°C for 45 seconds, annealing at a temperature listed in Table 1 for 45 seconds, extension at 72°C for 1 minute, termination at 72⁰C for 10 minutes, and soaking at 4°C.

9. The process according to claim 4, wherein the amplified product is detected using gel electophoresis.

0. The method for characterization and analysis of mammalian cells as claimed above exemplified herein substantially in the examples and figures.