All Things Stem Cell

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 there is great potential for using stem cells in regenerative therapies, there is still a ways to go before it can be considered a proven practice, although recent breakthroughs, and one specific trial in particular, makes it seem much closer. Recently, the first human tissue-engineered organ using stem cells was created and transplanted successfully into a patient. Other tissue regeneration efforts with stem cells have also recently made many breakthroughs, emphasizing the potential of using stem cells in future tissue transplants.

In the first reported instance of using stem cells to bioengineer a functional human organ, Paolo Macchiarini and his research group used a patient’s own stem cells to generate an airway, specifically a bronchus, and successfully grafted it into the patient to replace her damaged bronchus (See Figure 1). Macchiarini’s group bypassed the problem of immune rejection by using the patient’s own stem cells. Additionally, by combining a variety of bioengineering efforts, no synthetic parts were involved in the creation of the organ; it was made entirely of cadaveric and patient-derived tissues (Macchiarini et al., 2008; Hollander et al., 2009).

Figure 1. In order to create a patient-compatible replacement bronchus, Macchiarini’s group removed and decellularized a trachea from a cadaveric donor, grew cells removed from the patient on the trachea in a bioreactor, and then transplanted the bioengineered airway into the patient, successfully replacing their defective bronchus (Macchiarini et al., 2008).

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admin Bioengineering, Mesenchymal Stem Cells adult, clinical trials, mesenchymal, regenerative medicine

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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admin Cancer Stem Cells, Embryonic Stem Cells, Induced Pluripotent Stem Cells adult, cancer, embryonic, news, regenerative medicine

Cancer stem cells (CSCs), as their name implies, are stem cells that have been discovered to reside within cancerous tumors. Tumors are made up of a heterogeneous mixture of cells. Consequently, if the growth comes from a common origin it must be a cell, or cells, capable of becoming many different types of cells. This makes stem cells a very likely suspect as they, by definition, are able to give rise to a variety of cells. CSCs have been broadly defined as cells within a tumor that are able to self-renew, regenerating a population of multipotent CSCs, as well as differentiate into other cells, which can create the heterogeneity seen in tumors (Vermeulen et al., 2008).

Although the theory of cancer stem cells has been around since the 1970s (Hamburger and Salmon, 1977), recently it has gained a spotlight in the scientific community. The first functional identification of CSCs was in 1997 in acute myeloid leukemia (Bonnet and Dick, 1997). Researchers found that although there are many different populations of cells within a tumor, only one population has the ability to generate the tumor. This was determined by separating the populations from each other and engrafting them into an immuno-compromised (NOD/SCID) mouse; the population identified as CSCs was able to recreate the original tumor, including morphology and the specific differentiated cell types observed within the tumor (Vermeulen et al., 2008).

The different populations within a tumor can be separated and identified according to the proteins expressed (or produced) on the surface of a particular cell; cells expressing the same set of proteins are grouped into one population. Because such proteins are commonly used to identify and categorize cells, they are called cell markers. CSCs from the same tumor type usually have the same set of markers expressed, although the markers expressed can vary much more between CSCs from different tissues (Vermeulen et al., 2008). For example, breast cancer CSCs have been found to express a marker called CD44, but are distinct for also not expressing the marker CD24 (making this CSC population be labeled CD44⁺/CD24^-) (Al-Hajj et al., 2002). In comparison, pancreatic cancer CSCs express CD44, but also express CD24 (Li et al., 2007). Although there are differences like this in marker expression between CSCs from different tumor types, some markers are present in CSCs from many different types of tumors, such as CD44. CSCs from ovarian tumors (Zhang et al., 2008) and head and neck squamous cell carcinomas (Prince et al., 2006) have also been found to express CD44. Another major marker protein expressed in CSCs across tissue types is CD133; it is expressed by CSCs found in brain (Singh et al., 2003), prostate (Lang et al., 2008), colon (O’Brien et al., 2007), lung (Eramo et al., 2007), and hepatic (Suetsugu et al., 2006) tumors. For a more detailed summary of marker expression of CSCs from the different tumors they have been discovered in, see Table 1.

Table 1. Cancer Stem Cell Populations Detected in Different Cancerous Tumors (CSC Markers and Percent of the Total Tumor)

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admin Cancer Stem Cells adult, cancer, history

Virtually identical to human embryonic stem cells (hESCs) except for their origin of isolation, the recently created induced pluripotent stem cells (iPSCs) (Yu et al., 2007; Takahashi et al., 2007) hold much potential for use in regenerative therapies. iPSCs are cells that were originally from adult tissues, but have been forced to produce proteins that are thought to be essential for the pluripotency of human embryonic stem cells. By making cells express these embryonic stem cell proteins, adult cells can be created that look and act nearly identical to hESCs.

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.

The idea of reprogramming a cell from adult tissue into an embryonic-like, pluripotent cell existed long before the creation of iPSCs. In 1938, Hans Spemann showed that a nucleus from a fertilized salamander egg that had already undergone cell division several times could be implanted into a cell from a newly fertilized salamander egg that is enucleated (has had its nucleus removed) and create an entire adult salamander (Spemann, 1938). Consequently, Spemann’s work suggests that an embryonic nucleus remains totipotent, or is able to develop into any cell type of the adult body, even after several cell divisions. Due to technical difficulties, it was several years before researchers could repeat these experiments using older nuclei to see how long the nucleus retains its pluripotency. In the early 1950s, Robert Briggs and Thomas King repeated Spemann’s experiments using a species of leopard frog, Rana pipiens, first with a nucleus from young embryos (Briggs and King, 1952) then from older embryos (King and Briggs, 1954); both the younger and older implanted nuclei could still be reprogrammed by the enucleated host cell. However, they also observed that the older the donor nucleus was, the more difficult it was to reprogram it to a totipotent state. For years it was unclear whether the nucleus from a fully differentiated, adult cell could be completely reprogrammed, as conflicting results were published by different groups (Briggs and King, 1957; Fishberg et al., 1958; Gurdon and Byrne, 2003).

Although the studies done by Spemann, Briggs, and King used nuclei from embryos, their results are the basis for somatic cell nuclear transfer (SCNT). SCNT is a technique wherein 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 enucleated egg cell which can then be implanted into, and develop in, a surrogate mother, and potentially become an adult organism. The resultant organism is a clone of the animal that donated the nucleus. The first widely-accepted successful use of SCNT came with the creation of the sheep Dolly in 1997, the first cloned animal from an adult cell and the first cloned mammal (Wilmut et al., 1997). Since then, several other animals have been successfully cloned, though many problems still remain and there are low success rates (Wilmut et al., 1997; Wakayama et al., 1998; Solter, 1998; McKinnell and Di Bernardino, 1999; Gurdon and Byrne, 2003).

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admin Embryonic Stem Cells, Induced Pluripotent Stem Cells adult, embryonic, history, news