Flow Cytometry Essay

Custom Student Mr. Teacher ENG 1001-04 2 October 2016

Flow Cytometry

1. Introduction

1.1. Flow Cytometry – Definition

Flow cytometry is a powerful technique for the analysis of multiple parameters of individual cells within heterogeneous populations. Flow cytometers are used in a wide range of applications, such as immunophenotyping, cell counting and reporter gene (e.g. green fluorescence protein (GFP)) expression analysis [1]. This simultaneous parametric model analysis of the physical and/or chemical characteristics is obtained by passing thousands of cells per second through a laser beam and capturing the light of each cell as it passes through it.

The data gathered can be analyzed statistically by flow cytometer software to report cellular characteristics such as complexity, size, phenotype or viability, as well to purify populations of interest with Fluorescence-activated cell sorting (FACS) . This technology has a high number of applications, including molecular biology, immunology and in medicine (e.g. transplantation, tumor immunology and chemotherapy, genetics, and sperm sorting for sex pre-selection). The use of fluorescence tagged antibodies is useful in the field of molecular biology as they bind to specific antigens giving unique information about the cells being studied in the cytometer.

1.2. Principles of Flow Cytometry

Fig. 1 – Schematic view of the main components of the Cytometer. 1 – Fluidic system; 2 – Lasers; 3 – Optics; 4 – Detectors; 5 – Electronics and computer system; 6 – interrogation point. The Flow Cytometer is composed of several components; figure 1 shows a schematic representation of the interior of this equipment. The main components are: the fluidic system, which presents samples to the interrogation point and takes away the waste after that point; the lasers which produce a beam of light of a single wavelength and is directed onto a hydrodynamic focusing stream of fluid; the optic lenses that gather and direct the light; a number of detectors which are aimed at the point where the stream passes through the light beam: one in line with the light beam and several perpendicular to it; and finally the electronics and the peripheral computer system, responsible for the conversion of the electrical signal into digital data and to perform the necessary analyses. The interrogation point is the heart of the system. This is where the laser and the sample intersect and the optics collects the resulting scatter and fluorescence. Fig.2 – Schematic representation of the hydrodynamic focusing in flow cytometer.

For a good data collection, the particles or cells in study must pass through the laser beam one at a time. This is obtained by injecting the sample stream containing the cells into a flowing stream of sheath fluid or saline solution, as represented in figure 2. The sample stream becomes compressed and so narrow that roughly one cell passes through the channel at a time – this is called hydrodynamic focusing. Cytometers are able to detect particles between 1 and 15 microns in diameter. b


Fig. 3 – a: Laser beam passing through the particle and consequent FS (1) and SS (2); b: obscuration bar (1) and light sensor (2). Once the laser hits the cell, light will be refracted in all directions. Two different types of light scattering can be considered: Forward scattering (FS) and side scattering (SS) (figure 3a). As the light strikes the cells, it is scattered in the forward direction to the sensor – (FS). The magnitude of the FS light is proportional to the size of the cell, and this data can be used to quantify that parameter. To quantify this light most flow cytometers have a blocking bar (obscuration bar) (figure 3b). Thus, once the cells passes through the laser beam, light is scattered around this component and collected by the sensor.

This prevents any intense laser light from reaching the sensor. Small cells produce small amount of FS and big cells produce large amount of FS, so the magnitude pulse recorded for each cell is proportional to the cell size. Plotting a histogram of this information will put smaller cells in the left and larger cells in the right; In another way, once the laser beam hits the cells, light will be scattered in all directions, or in different words, in larger angles – SS. This SS at higher angles is caused by granularity and structural complexity inside the cells, it is focused through a lenses system and is collected by a separate sensor, usually located at 90 degrees from the laser’s path. Like it happens in the FS, the signals collected from the SS light detector can be plotted on one dimension histogram.

By studying only the forward scatter light, it is not possible to obtain a complete information about a population. For example, what appears to be a single population through FS analysis, can in fact be multiple populations that can be only separated by SS analysis and in a two dimensional plot of the resulting data. This is done through the use of two-dimensional dot or scatter plots. The peaks from the forward and side-scatter histograms correlate with the colored dots in the scatter plot, figure 4a. Fig.4 – a1: plots from FS and SS; a2: two dimensional dot or scattered plot, here it’s possible to see that the dots correlate to the peaks of the FS and SS plots; b: 2D scatter plot of blood, representing lymphocytes (low internal complexity), monocytes (medium sized cells and slightly more complex) and granulocytes (high internal complexity). As an example, we can take the scatter plot of a typical peripheral blood cell run, and see the results in a side scatter plot, using forward and side scatter data (figure 4b).

Here it is possible to observe lymphocytes, which are small cells with low internal complexity, monocytes which are medium-sized cells with slightly more internal complexity, and neutrophils and other granulocytes which are large cells that have a high internal complexity. When using flow cytometry it is possible to study cellular characteristics by labeling them specific antibodies with linked to fluorescent molecules. These antibodies will bind to the cell surface, or even to molecules inside the cells.

In the same way as described above, when laser light of the right wavelength strikes the fluorescent molecule, a fluorescent signal is emitted and detected by the sensor. In a solution with cells, some of them will be brighter than others, after passing through the laser beam, the fluorescent light will be translated into a voltage pulse proportional to the amount of fluorescence emitted, and then this information can be presented graphically.

Fig. 5 – Forward scatter threshold.

While collecting flow cytometry data, the use of a threshold (or discriminator or trigger) may be important. Indeed, since all the single particles that pass through the laser are counted, without the definition of a threshold, the dominant information that would appear would be concerning about minor particles such as platelets and debris. The purpose of creating a threshold is to set that a certain forward scatter pulse size must be exceeded for the instrument to collect the data. In figure 5, the area in the left of the red line represents the small cells and debris that are excluded from the analysis by the threshold. In this way, the major data that is presented by the flow cytometer corresponds to the cells of interest, although the small particles are still passing through the instrument, but being ignored.

2. Aplications of flow cytometry in stem cell analysis

Fig.6 – Pluripotent, embryonic stem cells can be isolated from the inner mass of the blastocyst. These stem cells can become any tissue in the body, excluding the placenta. Only the morula’s cells are totipotent, able to become all tissues and the placenta [2]. First of all, it is important to explain the concept of stem cells so we can understand how flow cytometry can be used as an important tool, for the study of stem cells. Stem cells are undifferentiated cells that have the ability of self-renewing themselves and that are capable of originating multiple cell lineages or more restricted progenitor populations, which in turn generate precursors and then fully mature cells. Stem cells may be found in the embryo and in adult tissues, contributing to tissue homeostasis by regenerating tissue after injury.

The capacity to differentiate into specialized cell types defines the potency of the stem cells. Thus, stem cells can be totipotent, having the capacity to specialize into all cell types, including the extraembrionic membrane and tissues; pluripotent, having the capacity to differentiate into cells from all the embryonic germ layers (endoderm, mesoderm and ectoderm); and multipotent that only differentiate into a limited range of cell types (figure 6) [2]. Other cells, named as progenitor cells, can divide a limited number of times before facing a change in their potency or undergoing differentiation [3]. In the adult body, it is likely that each tissue has a pool of stem cells that maintain their multipotency under strict growth control and can be mobilized to intervene in injury scenarios [4]. So the study of the molecular, cellular and development biology of embryonic and adult stem cells is a very powerful approach to understand the organization and function of complex tissues and organs [5].

Flow cytometry can be used for this purpose. Stem cells can be characterized namely by the expression of several transcription factors and cell surface proteins. As exposed above (section 1.2), the identification and quantification of expression of cellular antigens with fluorochrome-labeled monoclonal antibodies (“immunophenotyping”) is one of the most important applications of the flow cytometer. For flow cytometry analysis it is necessary to prepare single cell suspensions and to make them react with one or several immunoflurescent antibodies that will attach to the antigen whose expression is being analyzed.

For this work, two different scenarios were explored and will be presented. First, two different examples will be presented to illustrate how flow cytometry can be used for the characterization of the phenotype of both adult and embryonic stem cells. After that, it will also be illustrated how flow cytometry can be used to quantify the percentage of transfected stem cells and other types of mammalian cells after using respectively two different transfection techniques: lipofection and microporation.

3. Stem cell Phenotype characterization using flow cytometry

3.1 –Human Mesenchymal Stem Cells (hMSC)

Multipotent mesenchymal stem cells (MSC) are non-haematopoietic stromal cells that are capable of differentiating into distinctive end-stage cell types, such as bone, cartilage, muscle, bone marrow stroma, tendon/ligament, fat, dermis, and other connective tissues as diagrammed in Figure 7. MSC can secrete a wide spectrum of bioactive immunoregulatory molecules which play an important role in tissue regeneration [6]. Though not immortal, MSC have the ability to expand many-fold in culture, whilst retaining their growth and multi-lineage potential [7]. MSC of human origin (hMSC) are subject of intense and important study as they have useful clinical applications [8]. So, an important part of this study is to investigate whether or not hMSC maintain their characteristic phenotype after in vitro culture.

Presently, a set of standards is well established to define hMSC for both laboratory-based scientific investigations and for pre-clinical studies. First, MSC must be plastic-adherent when maintained in standard culture conditions using tissue culture flasks. Second, ≥95% of the MSC population must express cell surface markers CD105, CD90 and CD73. Third, the cells must be able to differentiate to osteoblasts, adipocytes and chondroblasts under standard in vitro differentiating conditions [9].

Fig. 7 – The Mesengenic Process diagram originated in the late 1980s as a hypothesis based on what was known about mesenchymal progenitors in embryos. The format was designed to mirror the lineage pathways of hematopoiesis with the bone lineage (Owen, 1985) on the left reflecting the state of knowledge, while the lineages at the right were largely unstudied. The original diagram appeared first in Caplan (1989).

CD105, CD90 and CD73 (CD stands for complement of differentiation) are antigen molecules expressed in the membrane of hMSC. At the molecular level, an antigen is characterized by its ability to be “bound” at the antigen-binding site of an antibody. In this work, flow cytometry was performed for hMSC immunophenotype characterization using antibodies that were labeled with a fluorescent dye, Phycoerythrin (PE). By virtue of its huge absorption coefficienct and almost perfect quantum efficiency PE it is one of the brightest dyes used today. It emits at about 570 nm, which corresponds to a green light detected in the flow cytometer [10].

3.1.1. Protocol

The protocol of the extracellular staining of cells used for flow cytometry is presented in annex I

3.1.2. Results and discussion

In this work, flow cytometry was used to evaluate the phenotype of hMSC after in vitro expansion in the presence of two different culture media: DMEM supplemented with 10% FBS (Fetal bovine serum) and the low serum commercial MesenPROTM. hMSC were cultured in the presence of these two culture media and afterwards the percentage of hMSC expressing CD105, CD90 and CD73 was evaluated by flow cytometry. There is a number of parameters that needs to be inputted to the flow cytometer, such us the voltage of the laser beam and the acquisition rate. After doing so, and using the dot-plot graphic corresponding to the physical characteristics of the cells (as explained in section 1.2), a gate of viable cells was selected, and their fluorescence was then quantified.

As previously explained, it is possible to draw this gate in the flow cytometer user interface so we can focus our study in the desired cells. Cell debris or other cells that are outside the gate Fig. 8 – Flow cytometry results for the expression of the extracellular markers (CD105, CD90 and CD73) in hMSC. From top to down CD 73, CD 90 and CD105. The histograms on the left show the results obtained with the medium DMEM supplemented with 10% FBS, and on the right, with low serum commercial MesenPROTM. will be excluded from the analysis.

An important aspect of flow cytometry analysis is the necessary use of a negative control run. For this purpose, isotypes of the monoclonal antibodies labeled with PE were used. In this way, it was possible to detect unspecific binding. In figure 8, the unspecific binding was represented by the first pick (1) of fluorescence in each graphic. After running the negative control, as depicted in (1), the fluorescence of the sample can also be measured. These values correspond to the second peak (2) of each graphic in figure 8.

As can be observed in figure 8, flow cytometer results shows that, under both culture conditions (DMEM supplemented with 10% FBS and MesenPROTM), more than 95% of hMSC express these characteristic markers. Thus, both culture media that are being used for the culture of the hMSC allow the correct phenotype to be expressed.

3.2. – Mouse Embryonic Stem Cells (mESC)

Embryonic stem (ES) cells are pluripotent cells that have the capacity of almost unlimited self-renewing, and they are able to differentiate into multiple cell types of the three embryonic germ layers: ectoderm, mesoderm and endoderm [11]. To date, most of the investigation has taken place using mouse embryonic stem cells (mES) or human embryonic stem (hES) cells. Both have the same essential characteristics, but they require very different culture conditions in order to maintain an undifferentiated state. The most important consideration is that, without optimal culture conditions, embryonic stem cells will rapidly differentiate [5]. Undifferentiated ES cells are evaluated as good material for applications in regenerative medicine, pharmacological and toxicological studies. So it is important to know and understand the factors affecting ES cell expansion and/or controlled differentiation in order to obtain a high number of cells for application in such areas [12].

In this work, mouse ES cells will be used to illustrate the application of flow cytometry for characterization of the phenotype of pluripotent stem cells. During in vitro expansion of mES cells it is crucial to maintain the undifferentiated state of these cells during long periods of time. This can be obtained by culturing the cells in serum-containing medium supplemented with leukemia inhibitory factor (LIF) [13]. However the use of serum imposes some limitations as it is a potential factor of pathogenic transmission. So, in this context, flow cytometry was used to evaluate the possibility of using serum-free culture medium to successfully support mES cells proliferation and maintenance of features during long periods of time [14]. Such as for the hMSC described in the section above, the expression of several typical mES cell markers, such as the intracellular markers Oct-4 and Nanog and the extracellular marker (SSEA-1) were evaluated by flow cytometry.

Oct-4 and Nanog are both transcription factors critically involved in the self-renewal of undifferentiated ES cells. Oct-4 expression must be closely regulated; too much or too little will actually cause differentiation of the cells, and is associated with an undifferentiated phenotype and tumors [15]. Overexpression of Nanog in mES cells causes them to self-renew in the absence of leukemia inhibitory factor. In the absence of Nanog, mES cells differentiate into visceral/parietal endoderm [16]. The antigen SSEA-1 (stage-specific embryonic antigen-1) is expressed at the morula stage in embryos. It is considered to function as a cell-cell interaction ligand in the compaction process. SSEA-1 is expressed also in undifferentiated F9 teratocarcinoma cells, which cease to express it after induction of differentiation [17].

3.2.1. Protocol

The protocol for the intracellular and extracellular staining of cells used for flow cytometry is presented in annex I

3.2.2. Results and discussion

mES cells can be expanded in serum-free conditions through activation of STAT-3 signaling by supplementation of exogenous LIF and induction of differentiation (ID) proteins by supplementation of bone morphogenic proteins (BMPs) [18]. For this work, the influence of initial cell density under serum-free conditions was studied in the expansion of mES cells. For that purpose, mES cells were plated at four different initial cell densities (104, 5×104, 105 and 5×105 cells/mL).

Fig. 9 – Pluripotent Mouse ES cells express high levels of transcription factors Oct-4 and Nanog, and cell surface marker SSEA-1 following expansion in serum-free medium, as assessed by flow cytometry analysis. Cells incubated only with secondary antibody, in the case of Oct-4 and Nanog, or with γ1-FITC isotype, for SSEA-1, were used as negative controls.

After expansion of mES cells, phenotypic analysis by flow cytometry revealed that, independently of the initial cell density, expanded cells expressed high levels of pluripotency markers, such as the cell surface marker SSEA-1 and the transcription factors Oct-4 and Nanog (figure 9). Indeed, almost 95% of the cells expressed Oct-4 and Nanog for all initial cell densities used. Concerning the SSEA-1 marker, the percentage of positive cells obtained was lower and between 70 and 94%. Overall, these results show that culture under serum-free conditions is able to maintain mouse ES cells pluripotency since proper signals are exogenously supplemented to the culture medium.

4. Study of transfection efficiency of hMSC using flow cytometry

Fig. 10 – Schematic representation of various transfection technologies and how they neutralize the negatively charged DNA. Transfection is the process of introducing genetic material into eukaryotic cells using non-viral methods [19]. Normally this technique involves the opening of transient pores in cell membrane, to allow the entering of nucleic acids material by using various chemical, lipid or physical methods. Thus, this gene transfer technology is a powerful tool to study gene function and protein expression in the context of a cell [20].

The transfected DNA and RNA are negatively charged molecules, so the critical problem of transfection is how to introduce these molecules into cells that also have negatively charged membranes (figure 10). This problem can be solved by using chemicals, like calcium phosphate and DEAE-dextran, or cationic lipid-based reagents that coat the DNA, neutralizing or even creating an overall positive charge in this molecule. Other physical methods, like microinjection or electroporation, simply punch the DNA through the membrane introducing it directly into the cytoplasm. The following section describes and discusses the use of lipid carriers for transfection of hMSC and the corresponding analysis of transfection efficiency through flow cytometry.

4.1. Transfection of hMSC using lipofectamine and corresponding flow cytometry analysis

Lipofectamine is a common commercial transfection reagent. It is used to introduce, which means to transfect, siRNA or plasmid DNA into in vitro cell cultures. Lipofectamine treatment alters the cellular plasma membrane, allowing nucleic acids to cross into the cytoplasm.

The mechanism of cationic lipid-mediated transfection originates from the basic structure of cationic lipids: a positively charged head group and one or two hydrocarbon chains. The positive surface charge of the liposomes mediates the interaction of the nucleic acid with the cell membrane, allowing for fusion of the liposome/nucleic acid (“transfection complex”) with the negatively charged cell membrane. The transfection complex is thought to enter the cell through endocytosis. Once inside the cell, the complex must escape the endosomal pathway and diffuse through the cytoplasm [21].

In this experiment, the purpose was the transfection of hMSC with the gene encoding for the GFP (green fluorescent protein) aiming at the optimization of in vitro transfection protocols for hMSC.

4.1.1. Protocol

The protocol used for the transfection of cells using lipofectamine is presented in annex I

4.1.2. Results and Discussion

The results of this experiment are presented in annex II. Different quantities of plasmid DNA and different quantities of lipofectamine were tested in order to optimize the transfection protocol. The results show that the percentage of hMSC expressing GFP varied between 14% and 20%. Thus, although lipofection is considered a gentle method, being able to maintain high levels of cell viability, in the case of these experiments, the transfection rate obtained was relatively low.

5. Study of human embryonic kidney 293 (HEK293) cells transfection efficiency using flow cytometry analysis

Until now, this work was focused in application of flow cytometry for adult and embryonic stem cell phenotype characterization and for evaluation of transfection efficiency studies using stem cells. In this section, however, flow cytometry was used to determine the transfection efficiency using a different mammalian cell line, the Human Embryonic Kidney (HEK) 293 cells. HEK293 cells are a specific cell line originally derived from human embryonic kydney cells grown in tissue culture. HEK293 cells are very easy to grow and transfect and have been widely-used in cell biology research for many years. They are also used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy. Due to their importance in these fields, in this work the transfection of HEK293 cells was optimized and the results were evaluated by using flow cytometry. For that purpose, the yellow fluorescence protein (YFP) was cloned in a vector alongside with the nuclear antigen EBNA (nuclear antigen) and transfected into HEK 293 cells.

This EBNA sequence allows the recombination of the plasmid DNA with the genetic material of the cells [22]. After transfection, HEK293 cells will express fluorescence that will be proportional to the amount of YFP present in the cytoplasm. Its excitation peak is 514nm and its emission peak is 527nm. YFP has reduced chloride sensitivity, faster maturation, and increased brightness (product of the extinction coefficient and quantum yield). Fig. 12 – Schematic representation of the cappilar used in microporation. Fig. 11- A diagram of the main components of an electroporator with cuvette loaded. In this particular case of HEK293, electroporation was selected as the transfection method.

Electroporation is based in the significant increase of the cell membrane electrical conductivity and permeability caused by an external electrical field (Figure 11). Thus, by electroporation it is possible to introduce genetic material into the cell, namely a piece of coding DNA, that will originate a mutagenesis in a specific gene. The technique requires fine-tuning and optimization of pulse duration and strength for each type of cell used. In addition, electroporation often requires more cells than chemical methods because of substantial cell death, and extensive optimization often is required to balance transfection efficiency and cell viability.

Modern electroporation instruments allow nucleic acid delivery to the nucleus and thus the successful transfer of DNA and RNA to primary and stem cells. In addition, the use of capillary instead of the cuvettes used in electroporation, allows microporation to be a more efficient technique. The gap size between the two electrodes is maximized and the surface area of electrode can be minimized compared to the cuvette type chamber, as shown in figure 12. By doing so, the transfection efficiency and cell viability is dramatically increased.

* Next, a schematic view of the experiment is presented next:

As exposed above, the process of microporation needs to be optimized for each cell type. So, in this experiment, several conditions were tested for transfection of HEK293 cells. Two different plasmids were tested: the pCEP4-YFP, with 11Kb of length, that was specifically designed for these types of cells, and the pBGH-YFP, with 4Kb length. For each plasmid, several voltages of microporation were used: 1000v, 1100v, 1200v, 1300v and 1400v. Finally the percentage of microporated viable cells was quantified by flow cytometry.

5.2.1. Protocol

The protocol used for the electroporation of HEK293 cells is presented in annex I

5.2.2. Results and Discussion

As performed for the other analysis in the previous sections, after gating live cells in the physical characteristics dot-plot, they are then analyzed concerning their fluorescence, as it is shown in the histogram of figure 14. In all three histograms it is possible to see a peak inside the M1 region representing the cells that are negative for the YFP expression. On the other hand, the M2 region contains the cells that emit fluorescence. Indeed, the FL1-H axis is a measure of the fluorescence emitted by the cells. The correspondent histogram statistics can also be obtained using a specific software. With this we can see the percentage of counted cells and also the percentace of gated cells in each region. a

Fig. 14 – The first histogram, a) represents the negative control run with cells that weren’t transfected. The second graphic, b) quantifies the fluorescence of the cells transfected with pCEP plasmid, using 1400V. The last graphic, c) quantifies the fluorescence of the cells transfected with the pBGH plasmid, using 1400V. On the right it is the statistical analysis of the each corresponding histogram. The first result presented corresponds to the negative control run (figure 14, a), a flow cytometer analysis of HEK293 cells that were not transfected in the microporator.

By observation of the histogram in figure 14a (control run), it was possible to conclude that until a level of fluorescence of 102, HEK293 cells are negative for expression of YFP (M1 region). In the same way, only after 102 fluorescence, cells will be positive for the expression of YFP (region M2). After running the negative control, HEK293 cells after microporation were analyzed by flow cytometry to determine the percentage of transfected cells as can be seen in the M2 region (figure 14).

When comparing the results obtained with the two different plasmids tested, the best results were achieved for the pCEP4-YFP plasmid, as it can be seen when comparing the percentage of cells in M2 regions in figure 14b and c (45% for pCEP4-YFP and 40% for pBGH-YFP). Normally, it is expected that a smaller plasmid (pBGH-YFP) will be more successfully transfected into a cell, since the metabolic burden caused by this plasmid will be lower. However, probably because pCEP4-YFP was specifically designed for HEK293 cells, a higher amount of YFP expression was obtained.

After observing the results obtained, it is possible to conclude that the percentage of viable transfection is relatively low. This may be attributed do to the fact cells do not support the electric pulse during the transfection, and they die as a consequence. Another point is that plasmid DNA may not be able to totally recombine with the genomic DNA of the cell, and thus cells will not emit fluorescence In conclusion, and as it was said before, the microporation technique requires optimization and study.

6. Conclusions

Flow cytometry is a versatile tool with enormous potential for the study of cells and particles. It can be used to determine many morphologic, molecular, biophysical and functional cellular characteristics and because of their unique analytical capabilities flow cytometry has become an integral part of the biotechnology research. The main goal of this work was to show some applications of flow cytometry in stem cell phenotype analysis and also as a tool for quantifying the transfection efficiency of stem cells and other mammalian cell types.

Flow cytometry is also used on a daily basis in hospitals and commercial laboratories for analysis of red cell, white cell, and platelet counts and to determine differential white cell counts. These clinical applications can be used to observe cellular aberrancies and assist in the therapeutic decisions as well as to help predicting clinical outcomes. In a near future, with more studies at the molecular level and with further refinements in the technology, opportunities to apply flow cytometry should be even more abundant.

7. Acknowledgment

Finally, I would like to thank the orientation of Doctor Margarida Diogo throughout all this work and all the collaborators at the BERG/IBB research lab.

8. References

[1] Roger S. Riley, Michael Idowu. Principles and Applications of Flow Cytometry. [2] Hans R. Schöler (2007). “The Potential of Stem Cells: An Inventory”. in Nikolaus Knoepffler, Dagmar Schipanski, and Stefan Lorenz Sorgner. Humanbiotechnology as Social Challenge. Ashgate Publishing, Ltd. pp. 28. [3] Ahmed, S. (2009). The Culture of Neural Stem Cells. Journal of Cellular Biochemistry 106:1–6 (2009). [4] Evans GS, Potten CS. 1991. Stem cells and the elixir of life. Bioessays 13:135–138. [5] Sanberg P. 2007. Neural stem cells for Parkinson’s disease: To protect and repair. Proc Natl Acad Sci USA 104:11869–11870. [5] Chambers I, Colby D, Robertson M, et al. (2003). “Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells”. Cell 113 (5): 643–55. [6] Arnold I. Caplan. Adult Mesenchymal Stem Cells for Tissue Engineering Versus Regenerative Medicine. Journal of cellular physiology. J. Cell. Physiol. 213: 341–347, 2007. [7] Mesenchymal Stem Cells: their Phenotype, Differentiation Capacity, Immunological Features and Potential for Homing. Giselle Chamberlain, James Fox, Brian Ashton and Jim Middleton. 10.1634/stemcells.2007-0197. [8] Dominici, M., Blanc, K Le, Mueller, I., Slaper-Cortenbach, I., Marini, Fc, Krause, Ds, Deans, Rj Keating, A., Prockop, Dj and Horwitz, Em (2006) ‘Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement’, Cytotherapy, 8:4, 315 — 317. [9] Dominici, M., Blanc, K Le, Mueller, I., Slaper-Cortenbach, I., Marini, Fc, Krause, Ds, Deans, Rj, Keating, A., Prockop, Dj and Horwitz, Em (2006) ‘Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement’, Cytotherapy, 8:4, 315 — 317. [10] Hardy, RR: Purification and coupling of fluorescent
proteins for use in flow cytometry. In: Handbook of Experimental Immunology, 4th ed. DM Weir, LA Herzenberg, C Blackwell, and LA Herzenberg, editors. Blackwell Scientific Publications, Boston, 1986, pp. 31.1-31.12. [11] Smith AG (2001) Embryo-derived stem cells of mice and men. Annu Rev Cell Dev Biol 17:435-462. [12] Hook L, O’Brien C, Allsopp T (2005) ES cell technology: nan introduction to genetic manipulation, differentiation and therapeutic cloning. Adv Drug Deliv Rev 57:1904-1917. [13] Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotent cells from mouse embryos. Nature 292(5819):154-156. [14] Wiles MV, Johansson BM (1999) Embryonic stem cell development in a chemically defined medium. Exp Cell Res 247:241-248. [15 Looijenga LH, Stoop H, de Leeuw HP, et al. (2003). “POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors”. Cancer Res. 63 (9): 2244–50. [16] itsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S (May 2003). “The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells”. Cell 113 (5): 631–42. [17] Kudo and Narimatsu (1995). The {beta}1,4-galactosyltransferase gene is post-transcriptionally regulated during differentiation of mouse F9 teratocarcinoma cells. Glycobiology vol 5 no 4 pp. 397-405, 1995 [18] Ying Q-L, Nichols J, Chambers I, Smith A (2003) BMP induction of Id proteins supresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115:281-292. [19] Graham FL, Smiley J, Russell WC, Nairn R (July 1977). “Characteristics of a human cell line transformed by DNA from human adenovirus type 5”. J. Gen. Virol. 36 (1): 59–74. [20] Promega. “Transfection – Protocols & Applications Guide”. www.promega.com [21] http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Transfection/Choosing-a-Transfection-Reagent.html [22] Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells

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