All Things Stem Cell

Induced pluripotent stem cells (iPSCs) have enormous potential for being used in tissue transplants and therapies. Why is this? It’s because iPSCs can be made using virtually any cells from a person’s body — such as skin or fat samples that wouldn’t be missed. (And since they’re the patient’s own cells, the immune system should not reject them in a transplant.) iPSCs are also pluripotent, which means they can be turned into nearly any cell type. This means that if a patient needs a certain type of retinal cell to treat an eye disease, a skin sample could be taken, turned into the desired retinal cells, and then transplanted into the patient’s eye. And this is exactly what happened for the first time last week.

Even though human iPSCs were first created in late 2007, it wasn’t until last week that they were used in a tissue transplant in a human for the first time. The person was a Japanese woman in her 70s, and the iPSC-derived cells were used to treat her age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Safety studies in mice and monkeys had been previously done to ensure the iPSCs would not cause tumors or be rejected by the immune system.
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One of the major hurdles that needs to be overcome in the field of regenerative medicine is the issue of immune rejection, or preventing a patient’s body from
rejecting a tissue transplant from a foreign donor. Consequently, researchers have increasingly focused on ways to regenerate damaged or diseased tissues in a patient by using the patient’s own tissues, which should not trigger an immune response. At this point in time, there are primarily two types of stem cells that hold the greatest promise for use in regenerative medicine where immune rejection is a significant concern: human induced pluripotent stem cells (iPSCs) and cells made through a process called somatic cell nuclear transfer (SCNT). This article will focus on recent SCNT improvements, but we’ll re-visit iPSCs briefly for comparison’s sake.

Applying Somatic Cell Nuclear Transfer in the Creation of Dolly the Cloned Sheep. Dolly the sheep was cloned through somatic cell nuclear transfer (SCNT). An adult cell from the mammary gland of a Finn-Dorset ewe acted as the nuclear donor; it was fused with an enucleated egg from a Scottish Blackface ewe, which acted as the cytoplasmic (or egg) donor. An electrical pulse acted to fuse the cells and activate the oocyte after injection into the surrogate mother ewe. A successfully implanted oocyte developed into the lamb Dolly, a clone of the nuclear donor, the Finn-Dorset ewe. SCNT may also be used to create patient-specific stem cells with great therapeutic potential.

Human induced pluripotent stem cells: The history and biology of human iPSCs were explored previously in “Induced Pluripotent Stem Cells: A New Stem Cell Line with a Long History.” In essence, iPSCs, which were first created with mouse cells in 2006 (Takahashi and Yamanaka, 2006) and then with human cells in 2007 (Yu et al., 2007; Takahashi et al., 2007), are adult cells that have been “reprogrammed” to an embryonic stem cell (ESC) state. This reprogramming is done by forcing adult cells to express proteins that are essential to the ESC identity (by transducing the adult cells with a retrovirus vector that contains the DNA for the key proteins). Consequently, human iPSCs look and behave nearly indistinguishably from hESCs. Like hESCs, iPSCs are pluripotent (they can become any cell type) and proliferate virtually indefinitely, both features which are important for use in regenerative medicine.

However, while great improvements have been made to make this technology closer to the clinic (such as multiple approaches to create iPSCs that do not have the reprogramming genes randomly integrated into their genomes [Yu et al., 2009; Zhou et al., 2009]), and it may someday be used to generate patient-specific ESC-like cells, the technology is not quite there yet. (Other similar technologies, such as “direct reprogramming,” are also being explored for the generation of patient-specific cells, but, again, this approach also has a ways to go.)

Somatic cell nuclear transfer: SCNT technology significantly predates iPSCs, and in many ways formed the basis for the idea of iPSCs. In SCNT, the nucleus from a somatic cell (an adult cell that is not a sperm or egg, i.e. not the gametes) is implanted into an egg, which already had its own nucleus removed. The egg amazingly reprograms the nucleus to become embryonic again; it’s been found that SCNT causes some 10,000 to 12,000 genes to be expressed (turned into protein) that are normally associated only with embryonic development (Niemann et al., 2008). The newly formed embryo (technically called a blastocyst) can then be implanted into a surrogate mother, and potentially become an adult organism. The organism is a clone of the animal that donated the nucleus. Although nuclear transfer studies have been conducted since the late 1930s (primarily in amphibians using nuclei donated from embryos, not adult tissues) (Spemann, 1938), it wasn’t until 1997 that the first widely-accepted successful use of SCNT was reported: Dolly the sheep was born, and she was the first cloned animal from an adult cell, and the first cloned mammal (Wilmut et al., 1997). Since Dolly, several other animals have been successfully cloned, though many problems still remain (the frequency of successful development is relatively low, as SCNT-derived embryos usually result in about 0 to 10% live births) (Wilmut et al., 1997; Wakayama et al., 1998; Solter, 1998; McKinnell and Di Bernardino, 1999; Gurdon and Byrne, 2003, Beyhan and Cibelli, 2008).

Therapeutic cloning: While SCNT has been long-explored for its ability to create cloned animals like Dolly and others (a practice called “reproductive cloning”), SCNT has other very appealing applications that do not involve the creation of an entire animal, such as “therapeutic cloning.” The goal of therapeutic cloning is to use SCNT technology to create patient-specific embryonic stem cells for medical therapies. These much sought-after cells are being labeled nuclear transfer stem cells (NTSCs), but they are essentially ESCs. While using SCNT to clone an entire animal has been fraught with developmental challenges, using SCNT to create NTSCs may be less difficult because it only requires a very early stage embryo, a blastocyst, to be formed (and NTSCs from a blastocyst may be less compromised by developmental abnormalities than an entire animal would be) (Beyhan and Cibelli, 2008). NTSCs have now been created from many different model animals.

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admin Embryonic Stem Cells, Induced Pluripotent Stem Cells, Reprogramming

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.

admin Embryonic Stem Cells, Induced Pluripotent Stem Cells, Reprogramming adult, cancer, developmental, embryonic, hematopoietic, history, news, potency, regenerative medicine

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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admin Embryonic Stem Cells, Induced Pluripotent Stem Cells embryonic, history, potency