All Things Stem Cell

A recently published paper showed that mice with colon cancer can be “vaccinated” with human embryonic stem cells and have a significant immune response against the cancer (Li et al., 2009). This study relates to a big hurdle that needs to be overcome in order to better fight cancer: immune tolerance. The immune system usually fails to detect and attack cancerous tumors, and consequently many cancer treatments are currently being developed that stimulate the immune system to fight back (e.g. the growing field of cancer vaccines).

Cancerous tumors and embryonic tissues have been found to share many of the same antigens, which are detected by the immune system through antibodies. This group of antigens is called oncofetal antigens. Consequently, animals can be vaccinated with embryonic tissues/cells (most recently done with human embryonic stem cells) and develop an immune response against cancer.

Interestingly, this state of immune tolerance is similar to what happens during pregnancy, and, more specifically, it’s been found that the body’s response to a tumor is very similar to its response to embryonic tissues. While much recent research has not been published in this area, there is actually a long history of studies that show: (1) there is a significant number of antigens shared between tumors and embryonic tissues (called “oncofetal antigens”) and, consequently, antibodies made against tumors can also recognize embryonic tissues, and vice versa; (2) pregnancy confers some immunity against cancer (accompanied by antibody production against oncofetal antigens), not only against its occurrence but also against its growth; (3) similar to pregnancy, an immune response against cancer can be generated by vaccinating animals with embryonic tissues. These studies and the recent re-visitation will be explored below (for a more detailed review, see Brewer et al., 2009).

The first published suggestion that tumors may have an embryonic nature came in the early 1800s (Muller, 1838). Tumors were suspected to be tissues that had been triggered to become embryonic-like again, and it is now generally accepted that tumors are indeed more “embryonic” than the tissues they are derived from, due to the re-expression of embryonic-related genes. By the late 1800s, researchers understood cancer enough to realize that they must better understand normal development in order to better combat cancerous tumors and their embryonic-like cells (Brewer et al., 2009). In the 1880s, these studies shifted focus; the field of immunology was born (from research conducted by Louis Pasteur, at the University of Strasbourg, and Robert Koch, as a medical officer in Poland) and many researchers focused on creating vaccines to cure diseases. Cancer was no exception.
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admin Cancer Stem Cells, Embryonic Stem Cells, Induced Pluripotent Stem Cells cancer, embryonic, history, news

A goal of regenerative medicine has been to be able to take any cell from a person’s body and turn it in to any other cell type that may be desired (such as insulin-producing beta-cells for treating diabetes, or creating neurons to treat a neurodegenerative disease). This would eliminate several donor-compatibility problems, and potentially eliminate the need for a donor (who isn’t the patient) altogether. In 2007, human induced pluripotent stem cells (iPSCs) were created and this goal seemed a bit closer (Yu et al., 2007; Takahashi et al., 2007). iPSCs are cells that can be take from adult tissue and “reprogrammed” into embryonic stem cell (ESC)-like cells. Because iPSCs are pluripotent, these cells can then differentiate into (or become) any cell type (for more information, see the All Things Stem Cell article on “Induced Pluripotent Stem Cells: A New Stem Cell Line with a Long History”).

But is it possible to get rid of the iPSC-middle man? Is it possible to take any cell in the adult body and directly reprogram it, skipping the iPSC state, into the final desired cell type? There have been several studies over the last few decades that show this is quite possible, though it still has a ways to go before it can be regularly used in the clinic.

Reprogramming of cells to a different cell type is usually done by either somatic cell nuclear transfer (SCNT) or by using transcription factors. This post will focus on work done with transcription factors (for more information on using SCNT, see the “Induced Pluripotent Stem Cells…” post). Transcription factors are expressed (or made) at different levels in different cell types, and control what genes are expressed in every cell, making sure, for example, that a liver cell remains a liver cell and does not become a neuron. A famous example of how transcription factor expression can be used to alter a cell’s identity is the creation of iPSCs, where adult cells were forced to express transcription factors normally expressed in ESCs, which made the adult cells express genes specific to ESCs, and consequently become nearly identical to ESCs.

There are many degrees of direct reprogramming that have been reported over the last few decades. Several progenitor cells, cells that appear to be committed to their fate but not yet fully differentiated, have been shown to be capable of dedifferentiating into a different cell type; this process is called transdetermination. However, in a few cases it has been shown that a fully differentiated cell can actually become a different cell type; this process is called transdifferentiation (Graf and Enver, 2009). Over the last few decades, much progress has been made in direct reprogramming with muscle, blood, the pancreas, and neurons.

Muscle

In the 1980s, the first reprogramming experiments using transcription factors took place. In 1987, a group reported using MyoD to make fibroblasts become muscle cells (Davis et al., 1987). Fibroblasts are cells important for wound healing (they secrete essential extracellular matrix proteins) and are common in connective tissues. The specific fibroblasts used were embryonic mouse fibroblasts. Because they were embryonic, this process is called transdetermination; the embryonic fibroblasts could probably differentiate more easily than adult fibroblasts (Graf and Enver, 2009). To convert the fibroblasts into muscle cells, the researchers transfected the fibroblasts with the cDNA of MyoD, forcing the cells to express MyoD (Davis et al., 1987). MyoD is normally only expressed in skeletal muscle, and it was later found to be a transcription factor involved in the differentiation of muscle cells and also a very early marker of muscle cell fate commitment.

Because of its success with the fibroblasts, MyoD was subsequently used in many other reprogramming studies to see what other cells it could make into muscle. It was found that while MyoD could indeed convert many different cell types into muscle, including fibroblasts in the dermal layer of skin, immature chondrocytes (cells in cartilage), smooth muscle, and retinal cells (Choi et al., 1990), MyoD could not turn any cell type into muscle; it was found incapable of making muscle out of hepatocytes (cells in the liver) (Schäfer et al., 1990).

Blood

In the 1990s, another key direct reprogramming factor was discovered, specifically involved in hematopoiesis. Hematopoiesis is the process by which the different types of blood cells are generated in the body (the term literally means “to make blood”). (For information on hematopoietic stem cells, see the All Things Stem Cell article “Hematopoietic Stem Cells: A Long History in Brief”). The central hematopoiesis-regulating factor discovered was the transcription factor GATA-1.

In 1995, a group reported that when GATA-1 was added to or removed from avian monocyte precursors, it could turn them into erythrocytes, megakaryocytes, and eosinophils (Kulessa et al., 1995). To understand the significance of these findings an inspection of hematopoiesis is required (see Figure). During hematopoiesis, hematopoietic stem cells (HSCs) (also called hemocytoblasts) give rise to all the different types of blood cells. Specifically, HSCs can first differentiate into either a common myeloid progenitor cell or a common lymphoid progenitor cell; either progenitor then further differentiates into specific blood cell types.

Direct Reprogramming in the Hematopoietic System. Several different transcription factors have been found that can directly reprogram one type of blood cell into another. Changing the expression levels of GATA-1 in monocytes (red) can make them differentiate into eosinophils, erythrocytes, or megakaryocytes. Making B-cells (B lymphocytes) express C/EBP transcription factors (blue) can cause them to differentiate into macrophages. Lastly, C/EBPs can also inhibit the function of the transcription factor Pax5; when Pax5 is deleted in B-cells they differentiate into T-cells (T lymphocytes), though they first dedifferentiate into a common lymphoid progenitor.

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While it is widely accepted that embryonic stem cells (ESCs) have the ability to become any type of cell, the molecular causes for this characteristic are still under much investigation, although one suspected player is chromatin. Recently, more evidence has been reported to support the important role of chromatin structure in maintaining an undifferentiated state in ESCs; the specific protein involved is called Chd1 (Gaspar-Maia et al., 2009).

DNA is condensed on histones, creating a structure called chromatin. (Left) A single DNA strand (formed by a sugar-phosphate backbone and nucleotide base-pairs). (Right) Chromatin is the complex formed by histones (green) and DNA (blue); the DNA can be tightly wrapped around the histones. (DNA bound to histones may be inaccessible to the transcription machinery, preventing the transcription of these genes, while unbound DNA allows space for the machinery and the genes may be transcribed.) Chd1 may function in ESCs to maintain chromatin in an open (euchromatin) state and potentially promote pluripotency in this way.

Chromatin structure plays an important role in regulating what genes are created, or expressed, in a given cell. In eukaryote organisms (almost all large organisms, such as animals, plants, and fungi, but not bacteria), DNA forms a complex with proteins that are called histones. This complex of DNA and histones is called chromatin (see figure). Histones act as spools for the DNA to be spun around, binding to DNA and packaging it into tightly coiled units (without histones, the long DNA strands would take up a very large amount of space). Whether the histones bind to the DNA or not can be regulated through chemical modification of the histones (they can be methylated or acetylated). When histones are bound to the DNA, the chromatin is in a condensed state (called heterochromatin) and the genes are not expressed because they cannot be accessed by the gene transcription machinery. However, when the histones are not bound to the DNA, the chromatin is extended (called euchromatin), and the DNA can be accessed and these genes can be expressed.

It was previously believed that embryonic stem cells had lots of open chromatin (euchromatin), but this was not a proven theory. A study on stem cells and gene expression (Efroni et al., 2008) reported that, globally but at low-levels, more genes in ESCs are actively turned into protein than are in differentiated cells. Additionally, proteins involved in changing chromatin structure and transcribing genes were expressed at relatively high levels in ESCs too. When the function of some proteins involved in chromatin-remodeling was changed, normal ESC proliferation and differentiation was also affected. Overall, Efroni et al. suggested that the differentiation of ESCs may correlate with a loss of active transcription of the cell genome.

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While embryonic stem cells are widely studied, a lesser known, but still significant, population of stem cells also resides within the early developing embryo: trophoblast stem cells (TSCs).

In brief, in most mammals the trophoblast is the part of the early embryo that later significantly contributes to the placenta of the fetus. The embryo and mother work together to create the placenta; while the trophoblast of the embryo becomes the chorion part of the placenta, the maternal uterine cells and surrounding blood vessels form the maternal placental components (Gilbert, 2003).

The placenta is the organ in mammals that connects the uterine wall to the developing fetus, bringing the two blood systems close together. The placenta allows the fetus to safely receive essential gases, such as oxygen, and nutrients from the mother. At the same time, it also lets the fetus expel waste through the mother’s kidneys. Additionally, the placenta releases essential pregnancy-related hormones and growth factors that, for example, let the uterus hold the fetus. Lastly, the placenta secretes immune response regulators to give the fetus immune protection against the mother (so that the fetus is not rejected by the mother’s immune system, as a tissue graft or organ transplant would be) (Rossant and Cross, 2001; Gilbert, 2003). Overall, the placenta plays a key role in early development; even small abnormalities in the placenta can lead to death of the fetus (Rossant and Cross, 2001).

Figure 1: The blastocyst is a hollow sphere made of approximately 150 cells and contains three distinct areas: the trophoblast, which is the surrounding outer layer that contains the trophoblast stem cells and later becomes the placenta, the blastocoel, which is a fluid-filled cavity within the blastocyst, and the inner cell mass, also known as the embryoblast, which can become the embryo proper, or fetus, and is where human embryonic stem cells are isolated from. When the late blastocyst is implanted in the uterine wall, at day 7 or 8 in human development, the trophoblast stem cells (in the trophoblast) quickly differentiate to form cells required for a firm implantation and, later, for the placenta.

While TSCs give rise to the placenta, these stem cells establish their identity long before the placenta develops; their fate is determined during the early embryo. Soon after the egg and sperm join during fertilization, the resultant zygote (fertilized egg cell) starts undergoing cell division. The resulting cells continue to undergo synchronous cell division. When the embryo is at the 16-cell stage (called a morula), it is a solid sphere of cells and already the precursors of the trophoblast cells are defined; the external, relatively larger cells mostly become the trophoblast cells. By the 64-cell stage, these cells’ fates are set; while the trophoblast will become the placenta, the other cells in the embryo can become the fetus. In mammalian development, this is the first differentiation event (Rossant and Cross, 2001; Gilbert, 2003).

A few cell divisions later, the trophoblast contributes to significant cellular rearrangements in the embryo which make it enter the blastocyst stage (see Figure 1). The blastocyst, which contains approximately 150 cells, is made up of three main parts: the blastocoel (an internal, fluid-filled cavity), the inner cell mass (ICM), and the trophoblast. When the embryo was a morula, the surrounding trophoblast precursors caused fluid to be secreted into the morula (utilizing sodium pumps in the trophoblast cell membranes); this secretion created the blastocoel cavity. The ICM is a cluster of cells inside the blastocyst that will later become the adult organism; human embryonic stem cells can be derived from the ICM, as was previously discussed. Lastly, the trophoblast is a monolayer of cells, specifically polarized epithelial cells, which surround the blastocoel and ICM, similar to their future role of surrounding the fetus as its placenta (Rossant and Cross, 2001; Gilbert, 2003).

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Recently, many papers have come out that highlight connections between cancer and induced pluripotent stem cells (iPSCs), the latter of which was discussed previously. These papers hold many implications for not only iPSCs, but for our understanding of cancer as well. Additionally, these papers should not at all be thought of as invalidating the importance of iPSCs for studying and treating future therapies, but they should help us better understand what iPSCs are and how to use them appropriately.

The most recent and most publicized link between iPSCs and cancer is p53. p53, also known as protein 53 (53 referring to its molecular mass), is a well-studied protein whose normal function is important in preventing cancer. Though p53 has many different roles, they are quite related. In essence, the job of p53 is to make sure the cell does not accumulate DNA damage, or DNA mutations, which could eventually make the cell cancerous. When a cell has its DNA damaged, often from external stresses, p53 stops the normal cell cycle to fix the DNA damage. If the damage is too great to repair, p53 can prevent the cell from dividing, which would create more damaged cells; p53 initiates programmed cell death, or apoptosis. The potential tumor cell dies. Overall, p53 functions as a “tumor suppressor” to prevent abnormal cells from occurring and multiplying into a cancer (Vazquez et al., 2008). Consequently, it has been found that p53 is mutated in approximately 50% of all human tumors, and other tumors may have mutations in the pathway regulating p53 activity (Vazquez et al., 2008). p53 is therefore well-studied as an oncogene, or a gene that when not functioning normally can contribute to a normal cell becoming cancerous.

So what does p53 have to do with iPSCs? One recently discovered connection is with the generation of iPSCs. Recently, many research groups discovered that when p53 is deleted from, or damaged in, their cells, they could more easily become iPSCs (Hong et al., 2009; Kawamura et al., 2009; Utikal et al., 2009; Li et al., 2009; Zhao et al., 2008). As posted earlier, iPSCs are cells that were originally from adult tissues, but have been “reprogrammed” to be pluripotent stem cells, or stem cells able to become all the adult cells of the body, looking and functioning nearly identical to human embryonic stem cells (hESCs) (Takahashi et al., 2007; Yu et al., 2007).

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