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Applying a "Double-Feature" Promoter To Identify Cardiomyocytes Differentiated From HESC

Embryonic Stem Cells/Induced Pluripotent Stem Cells

 

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Applying a "Double-Feature" Promoter To Identify Cardiomyocytes Differentiated From Human Embryonic Stem Cells Following Transposon-Based Gene Delivery

Tamás I. Orbán, Ágota Apáti, Andrea Németh1, Nóra Varga1, Virág Krizsik1, Anita Schamberger1, Kornélia Szebényi1, Zsuzsa Erdei1, György Várady1, Éva Karászi1, László Homolya1, Katalin Német1, Elen Gócza2, Csaba Miskey3, Lajos Mátés3, Zoltán Ivics3, Zsuzsanna Izsvák3, Balázs Sarkadi1*

1Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, Budapest, Hungary
2Genetic Modification Program Group, Agricultural Biotechnology Center, Gödöllalt, Hungary
3Mobile DNA Group, Max-Delbrück Center for Molecular Medicine, Berlin, Germany

email: Balázs Sarkadi (sarkadi@biomembrane.hu)

*Correspondence to Balázs Sarkadi, Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, Dioszegi u. 64., Budapest, H-1113, Hungary

altAuthor contributions: T.I.O.: Conception and design; collection and assembly of data; data analysis and interpretation; manuscript writing; Á.A.: Conception and design; collection and assembly of data; data analysis and interpretation; manuscript writing; A.N.: Collection and assembly of data; N.V.: Collection and assembly of data; V.K.: Collection and assembly of data; A.S.: Collection and assembly of data; K.S.: Collection and assembly of data; Z.E.: Collection and assembly of data; G.V.: Collection and interpretation of data; É.K.: Collection of data; L.H.: Interpretation of data; K.N.: Collection and interpretation of data; E.G.: Interpretation of data; C.M.: Data analysis; L.M.: Interpretation of data; Z.Ivics Data analysis; manuscript writing; Z.Izsvák: Data analysis and interpretation of data; B.S.: Conception and design; financial support; collection and assembly of data; data analysis and interpretation; manuscript writing; final approval of manuscript.
altFirst published online in STEM CELLS Express February 20, 2009.
§Tamás I. Orbán and Ágota Apáti contributed equally to this work.
Phone/Fax: +36 1 372 4353

Funded by:

  • EU FP6-INTHER; Grant Number: LSHB-CT-2005018961
  • OTKA; Grant Number: AT 048986, NK72057, NKFP-1A-060/2004, ETT 405/2006
  • KKK grants

 


Keywords

Sleeping Beauty transposon • human embryonic stem cells • CAG promoter • altdouble-featurealt promoter • cardiomyocytes • lentiviral gene delivery

 


Abstract

Human embryonic stem (HuES) cells represent a new potential tool for cell- and gene-therapy applications. However, these approaches require the development of efficient, stable gene delivery, and proper progenitor cell and tissue separation methods. In HuES cell lines we have generated stable, EGFP-expressing clones using a transposon-based (Sleeping Beauty) system. This method yielded high percentage of transgene integration and expression. Similarly to a lentiviral expression system, both the undifferentiated state and the differentiation pattern of the HuES cells were preserved. By using the CAG promoter, in contrast to several other constitutive promoter sequences (such as CMV, EF1alt, or PGK), an exceptionally high EGFP expression was observed in differentiated cardiomyocytes. This phenomenon was independent of the transgene sequence, methods of gene delivery, copy number, and the integration sites. This altdouble-featurealt promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifically labeling differentiated cardiomyocytes, was assessed by transcriptional profiling. We found a positive correlation between CAG promoter-driven EGFP transcription and expression of cardiomyocyte-specific genes. Our experiments indicate an efficient applicability of transposon-based gene delivery into HuES cells, and provide a novel approach to identify differentiated tissues by exploiting a non-typical behavior of a constitutively active promoter, thereby avoiding invasive drug selection methods.

 

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Stem Cells and Diseases

The Promise of Stem Cells

Studying stem cells will help us understand how they transform into the dazzling array of specialized cells that make us what we are. Some of the most serious medical conditions, such as cancer and birth defects, are due to problems that occur somewhere in this process. A better understanding of normal cell development will allow us to understand and perhaps correct the errors that cause these medical conditions.

Another potential application of stem cells is making cells and tissues for medical therapies. Today, donated organs and tissues are often used to replace those that are diseased or destroyed. Unfortunately, the number of people needing a transplant far exceeds the number of organs available for transplantation. Pluripotent stem cells offer the possibility of a renewable source of replacement cells and tissues to treat a myriad of diseases, conditions, and disabilities including Parkinson's and Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis and rheumatoid arthritis.

Have Human Embryonic Stem Cells Successfully Treated Any Human Diseases?

Scientists have been able to do experiments with human embryonic stem cells (hESC) only since 1998, when a group led by Dr. James Thompson at the University of Wisconsin developed a technique to isolate and grow the cells. Moreover, Federal funds to support hESC research have been available since only August 9, 2001, when President Bush announced his decision on Federal funding for hESC research. Because many academic researchers rely on Federal funds to support their laboratories, they are just beginning to learn how to grow and use the cells. Thus, although hESC are thought to offer potential cures and therapies for many devastating diseases, research using them is still in its early stages.

Adult stem cells, such as blood-forming stem cells in bone marrow (called Hematopoietic Stem Cells, or HSCs), are currently the only type of stem cell commonly used to treat human diseases. Doctors have been transferring HSCs in bone marrow transplants for over 40 years. More advanced techniques of collecting, or "harvesting," HSCs are now used in order to treat leukemia, lymphoma and several inherited blood disorders.

The clinical potential of adult stem cells has also been demonstrated in the treatment of other human diseases that include diabetes and advanced kidney cancer. However, these newer uses have involved studies with a very limited number of patients.

 

Stem Cells 101

What are Stem Cell Lines?

Stem cell lines are stem cells that have been isolated from tissue or blood and held in liquid Culture Medium under conditions designed to support their growth and Proliferation. Under the correct conditions this proliferation enables substantial expansion of the cell numbers. Following expansion, the stem cell cultures can be harvested, divided into vials and preserved at ultra-low temperatures. This stock of frozen cells is called a cell bank and the freezing process is a crucial stage which enables the cell bank to be stored in a viable and stable state until required. The cells can be thawed and re-cultured for research or therapy. Holding the cells in suspended animation this way also enables extensive quality control and safety testing to be performed before the cells are approved for use. In some cases, such as embryonic stem cells the cultures appear to have the capacity to expand indefinitely, without changing. Such cell cultures are called stem cell lines.

 

Stem Cells 101

 

Embryonic Stem Cells Developments

A major development in research came in May 2003, when researchers announced that they had successfully used Embryonic stem cells to produce human egg cells. These egg cells could potentially be used in turn to produce new Stem Cells. If research and testing proves that artificially created egg cells could be a viable source for Embryonic stem cells, they noted, then this would remove the necessity of starting a new Embryonic stem cell line with the destruction of a Blastocyst. Thus, the controversy over donating human egg cells and blastocysts could potentially be resolved, though a Blastocyst would still be required to start each cycle.

 

The online edition of Nature Medicine published a study on January 23, 2005 which stated that the Human Embryonic Stem CellsCulture Medium used to grow the cells, for example, mouse cells and other animal cells. The nonhuman cell-surface sialic acid can compromise the potential uses of the Embryonic stem cells in humans, according to scientists at the University of California, San Diego.

A study was published in the online edition of Lancet Medical Journal on March 8, 2005 that detailed information about a new stem-cell line which was derived from human embryos under completely cell- and serum-free conditions. This event is significant because exposure of existing human embryonic stem-cell lines to live animal cells and serum risks contamination with pathogens that could lead to human health risks. After more than 6 months of Undifferentiated Proliferation, these cells retained the potential to form derivatives of all three embryonic Germ layers both In vitro and in teratomas. These properties were also successfully maintained (for more than 30 passages) with the established stem-cell lines. (Lancet Medical Journal)

Recently, in California , researchers have injected Embryonic stem cells into mice as they developed in the womb. Upon maturing, it was found that some of the human ESCs had survived and two months after injection, the researchers found that the ESCs had undertaken ?the characteristics of mouse cells?

Scientists in Australia have grown human prostate in mice using Embryonic stem cells. In a world first, the scientists combined human Embryonic stem cells with mouse prostate cells, and used a mouse as the host to grow the human prostate. The researchers were able to show that it was also functioning as a human prostate. Doctors world wide would now be able to use this prostate as a model for studying prostate cancer and disease, and to produce future drugs. available for federally funded research are contaminated with nonhuman molecules from the

 

Stem Cells 101

 

Embryonic Stem Cells History

Embryonic stem cells were first derived from mouse embryos in 1981 by two independent research groups (Evans & Kaufman and Martin). The breakthrough in embryonic stem cell research came in November 1998 when a group led by James Thomson at the University of Wisconsin-Madison first developed a technique to isolate and grow the cells derived from human blastocysts. Normally, Blastocyst-stage embryos that are left over after successful In Vitro Fertilization would not be used but be destroyed.

 

Scientists are only allowed to use these discarded blastocysts after assessment by specialized committees that thoroughly check the research goals of these scientists. Of course, scientific research using those blastocysts may be conducted when it contributes to a better understanding of how to generate cells and tissues that can cure a patient?s disease or be used to treat severe injuries. Currently, in the United States , scientific research is limited by a ban on the use of federal funds for research with Embryonic stem cells derived after August 9th, 2001.

Embryonic stem cell researchers are currently attempting to grow the cells in the laboratory (i.e. in culture flasks: ?In Vitro?) beyond the first stages of cell development. It is important to make sure the Embryonic stem cells are fully differentiated into the desired cell type (i.e. tissue) before they are transplanted into the patient, as Undifferentiated Embryonic stem cells may develop into a tumor after transplantation. Further more, scientists are trying to develop techniques to prevent rejection of implanted cells by the patient (i.e. host-versus-graft response).

One of the possibilities to prevent rejection is by creating embryonic stem cell clones that are genetically identical to the patient. This can be achieved by fusing an egg cell, the nucleus (containing the genetic material: DNA) of which is removed, with a patient?s cell. The fused cell produced (containing only the DNA of the patient) is allowed to grow to the size of a few tens of cells, and Stem Cells are then extracted. Because they are genetically compatible with the patient, the patient?s immune system will not reject differentiated cells derived from these Embryonic stem cells. More commonly, they are obtained for research purposes from uncloned blastocysts, such as those discarded from In vitro Fertilization clinics. Such cells might be rejected if transplanted into a patient, as they do not contain identical genetic information. A possible solution for this is to derive as many well-characterized embryonic stem cell lines from different genetic and ethnic backgrounds and use the cell line that is most similar to the patient; treatment can then be tailored to the patient, minimizing the risk of rejection.

 

Stem Cells 101

Embryonic Stem Cells Research

Embryonic stem cells (ESCs) are Stem Cells derived from inner mass cells of a human Embryo (sometimes called a Blastocyst, which is an early stage Embryo - approximately 1 week old in humans - consisting of 50-150 cells). Embryonic stem cells are Totipotent, meaning they are able to grow (i.e. differentiate) into all derivatives of the three primary Germ layers: Ectoderm, Endoderm and Mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body as long as they are specified to do so. This characteristic property distinguishes Embryonic stem cells from adult Stem Cells or progenitor cells, the latter two of which only have the capacity to form a limited number of different cell types. Because of their unique combined abilities of unlimited expansion and pluripotency, Embryonic stem cells potentially are the ultimate source for Regenerative medicine and tissue replacement after injury or disease. To date, no medical treatments have been successfully derived from embryonic stem cell research as the first Human Embryonic Stem Cell Line has only been reported in 1998.

 

Stem Cells 101

 

Definition

Research using Embryonic stem cells remains at the zenith of stem cell science because, unlike somatic cells, Embryonic stem cells are Pluripotent. However, research using Stem Cells derived from the human Embryo is still in the basic research phase, as these Stem Cells were first isolated in 1998 (at least for humans), whereas adult Stem Cells have been studied since the 1960s. Research with Embryonic stem cells derived from humans is controversial because, in order to start a stem cell ?line? or lineage, the destruction of a human Embryo and/or Therapeutic Cloning is usually required. Some believe this to be a slippery slope to Reproductive Cloning and tantamount to the objectification of a potential human being.

In an attempt to overcome these moral, political and ethical hurdles, medical researchers have been experimenting with alternative techniques of obtaining Embryonic stem cells by extraction, which does not involve Cloning and/or the destruction of a human Embryo. Cancer Stem Cells arising through malignant transformation of adult Stem Cells are proposed to be the source of some or all tumors and cause metastasis and relapse of the disease. The stem cell origin of leukemias is well established.

The role of Stem Cells in other tumors is under intensive investigation. Cord Blood Stem Cells are derived from the blood of the placenta and umbilical cord after birth. Since 1988 these Cord blood stem cells have been used to treat Gunther?s disease, Hunter syndrome, Hurler syndrome, Acute lymphocytic leukemia and many more problems occurring mostly in children. Umbilical cord blood use has become so common that there are now umbilical cord blood banks that accept donations from parents. It is collected by removing the umbilical cord, cleansing it and withdrawing blood from the umbilical vein.

This blood is then immediately analyzed for infectious agents and the tissue-type is determined. The cord blood is processed and depleted of red blood cells before being stored in liquid nitrogen for later use, at which point it is thawed, washed of the cryoprotectant, and injected through a vein of the patient. This kind of treatment, where the Stem Cells are collected from another donor, is called allogeneic treatment. When the cells are collected from the same patient on whom they will be used, it is called autologous and when collected from identical individuals (i.e. homozygous twin), it is referred to as syngeneic.

 

Stem Cells 101

Sources Stem Cell Primer

Stem Cells are also categorized according to their source, as either adult, embryonic, cancer or Cord Blood Stem Cells.

 

Adult Stem Cells are Undifferentiated cells found among differentiated cells of a specific tissue and are mostly Multipotent cells. They are more accurately called somatic (Greek σωμα sōma = body) Stem Cells, because they need not come from adults but can also come from children or umbilical cords. Particularly interesting are adult Stem Cells termed "spore-like cells". They are present in all tissues[2] and seem to survive long time periods and harsh conditions.

Embryonic stem cells are cultured cells obtained from the Undifferentiated inner mass cells of an early stage human Embryo (sometimes called a Blastocyst, which is an Embryo that is between 50 to 150 cells). Embryonic stem cell research is "thought to have much greater developmental potential than adult Stem Cells," according to the National Institutes of Health.[3] Research using Embryonic stem cells remains at the zenith of stem cell science because, unlike somatic cells, Embryonic stem cells are Pluripotent. However, research using Stem Cells derived from the human Embryo is still in the basic research phase, as these Stem Cells were first isolated in 1998 (at least for humans), whereas adult Stem Cells have been studied since the 1960s.[4] Research with Embryonic stem cells derived from humans is controversial because, in order to start a stem cell 'line' or lineage, the destruction of a human Embryo and/or Therapeutic Cloning is usually required. Some believe this to be a slippery slope to Reproductive Cloning and tantamount to the objectification of a potential human being. In an attempt to overcome these moral, political and ethical hurdles, medical researchers have been experimenting with alternative techniques of obtaining Embryonic stem cells by extraction, which does not involve Cloning and/or the destruction of a human Embryo.

Cancer Stem Cells arising through malignant transformation of adult Stem Cells are proposed to be the source of some or all tumors and cause metastasis and relapse of the disease.[5] The stem cell origin of leukemias is well established[6]. The role of Stem Cells in other tumors is under intensive investigation.

Cord blood stem cells are derived from the blood of the placenta and umbilical cord after birth. Since 1988 these Cord blood stem cells have been used to treat Gunther's disease, Hunter syndrome, Hurler syndrome, Acute lymphocytic leukemia and many more problems occurring mostly in children. Umbilical cord blood use has become so common that there are now umbilical cord blood banks that accept donations from parents. It is collected by removing the umbilical cord, cleansing it and withdrawing blood from the umbilical vein. This blood is then immediately analyzed for infectious agents and the tissue-type is determined. The cord blood is processed and depleted of red blood cells before being stored in liquid nitrogen for later use, at which point it is thawed, washed of the cryoprotectant, and injected through a vein of the patient. This kind of treatment, where the Stem Cells are collected from another donor, is called allogeneic treatment. When the cells are collected from the same patient on whom they will be used, it is called autologous and when collected from identical individuals (i.e. homozygous twin), it is referred to as syngeneic.

 

Stem Cells 101

 

Potency Types

Stem cell potency specifies the ameliorative potential of the cell type.

Totipotent Stem Cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg cell are also Totipotent. These cells can differentiate into any type of cell without exception.

Pluripotent Stem Cells are the descendants of Totipotent cells and can differentiate into any cell type except for Totipotent Stem Cells.

Multipotent Stem Cells can produce only cells of a closely related family of cells (e.g. hematopoeietic Stem Cells differentiate into red blood cells, white blood cells, platelets etc.).

Unipotent cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-Stem Cells.

 

Stem Cells 101

 

What Are The Potential Uses Of Human Stem Cells And The Obstacles That Must Be Overcome Before These

There are many ways in which human stem cells can be used in basic research and in clinical research. However, there are many technical hurdles between the promise of stem cells and the realization of these uses, which will only be overcome by continued intensive stem cell research.

 

Studies of human embryonic stem cells may yield information about the complex events that occur during human development. A primary goal of this work is to identify how undifferentiated stem cells become differentiated. Scientists know that turning genes on and off is central to this process. Some of the most serious medical conditions, such as cancer and birth defects, are due to abnormal cell division and differentiation. A better understanding of the genetic and molecular controls of these processes may yield information about how such diseases arise and suggest new strategies for therapy. A significant hurdle to this use and most uses of stem cells is that scientists do not yet fully understand the signals that turn specific genes on and off to influence the differentiation of the stem cell.

Human stem cells could also be used to test new drugs. For example, new medications could be tested for safety on differentiated cells generated from human pluripotent cell lines. Other kinds of cell lines are already used in this way. Cancer cell lines, for example, are used to screen potential anti-tumor drugs. But, the availability of pluripotent stem cells would allow drug testing in a wider range of cell types. However, to screen drugs effectively, the conditions must be identical when comparing different drugs. Therefore, scientists will have to be able to precisely control the differentiation of stem cells into the specific cell type on which drugs will be tested. Current knowledge of the signals controlling differentiation fall well short of being able to mimic these conditions precisely to consistently have identical differentiated cells for each drug being tested.

Perhaps the most important potential application of human stem cells is the generation of cells and tissues that could be used for cell-based therapies. Today, donated organs and tissues are often used to replace ailing or destroyed tissue, but the need for transplantable tissues and organs far outweighs the available supply. Stem cells, directed to differentiate into specific cell types, offer the possibility of a renewable source of replacement cells and tissues to treat diseases including Parkinson's and Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis.

For example, it may become possible to generate healthy heart muscle cells in the laboratory and then transplant those cells into patients with chronic heart disease. Preliminary research in mice and other animals indicates that bone marrow stem cells, transplanted into a damaged heart, can generate heart muscle cells and successfully repopulate the heart tissue. Other recent studies in cell culture systems indicate that it may be possible to direct the differentiation of embryonic stem cells or adult bone marrow cells into heart muscle cells (Figure 4).

In people who suffer from type I diabetes, the cells of the pancreas that normally produce insulin are destroyed by the patient's own immune system. New studies indicate that it may be possible to direct the differentiation of human embryonic stem cells in cell culture to form insulin-producing cells that eventually could be used in transplantation therapy for diabetics.

To realize the promise of novel cell-based therapies for such pervasive and debilitating diseases, scientists must be able to easily and reproducibly manipulate stem cells so that they possess the necessary characteristics for successful differentiation, transplantation and engraftment. The following is a list of steps in successful cell-based treatments that scientists will have to learn to precisely control to bring such treatments to the clinic. To be useful for transplant purposes, stem cells must be reproducibly made to:

  • Proliferate extensively and generate sufficient quantities of tissue.

  • Differentiate into the desired cell type(s).

  • Survive in the recipient after transplant.

  • Integrate into the surrounding tissue after transplant.

  • Function appropriately for the duration of the recipient's life.

  • Avoid harming the recipient in any way.

Also, to avoid the problem of immune rejection, scientists are experimenting with different research strategies to generate tissues that will not be rejected.

To summarize, the promise of stem cell therapies is an exciting one, but significant technical hurdles remain that will only be overcome through years of intensive research.

 

Stem Cells 101

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