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 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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admin Trophoblast Stem Cells developmental, embryonic, 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

Salamanders have the amazing ability to re-grow a limb after it has been cut off. It is thought that by better understanding this regenerative ability, researchers will be able to apply this knowledge to humans and improve wound healing. Recently it was reported that salamander limb regeneration may occur in a different way than was previously thought; in short, the severed limb may not need pluripotent stem cells to regenerate, as was believed, but only multipotent or unipotent stem cells, stem cells with relatively restricted fates.

In salamanders, when a limb is severed the resultant limb bud undergoes a distinct process to regenerate the lost limb. The epithelial layer quickly spreads across the amputation site, closing the wound within 24 hours (Mescher, 1996). This epithelial layer thickens and becomes what is referred to as the wound epithelium (WE). As the immune system responds to the injury, macrophages and neutrophils arrive to clean up the wound site beneath the WE. The existing injured tissues and cells are broken down as well as the extracellular matrix, which is made up of proteins that surround cells to hold them together and stimulate normal cellular functions. It was thought that at this time in the regenerative process other resident cells below the WE become multipotent mesenchymal stem cells (MSCs) (see Figure). These eventually form a mass of MSCs called a blastema (Mescher, 1996; Brockes and Kumar, 2005). The blastema was thought to contain a homogenous group of pluripotent stem cells that had “dedifferentiated” or “redifferentiated,” meaning they had reverted back from their committed fates to function as very potent stem cells in order to recreate the limb. The WE stimulates the cells in the blastema to proliferate, making new cells and extracellular matrix, though more than is required for simple repair; the WE signals the blastema cells to regenerate the entire lost limb (Mescher, 1996; Kragl et al., 2009).

Limb regeneration in the salamander after limb amputation (time course going from the top down). Shortly after the limb is amputated, the epithelium layer covers the exposed limb bud, forming the wound epithelium (WE). A group of stem cells collects below this layer, forming the blastema (at the tip of the bud). The WE signals the stem cells below it to rebuild the limb, recreating the limb from the point of injury out towards the hand. The final regenerated limb is indistinguishable from the original.

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admin Mesenchymal Stem Cells, Non-Mammalian Stem Cells history, news, regenerative medicine