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

Often the feasibility of using stem cells for regenerative therapies is limited by two factors: obtaining a significant number of cells and doing so in a relatively noninvasive manner. Because our bodies freely shed a limited and select number of cells, many stem cell types must be obtained using a rather invasive procedure. However, around the beginning of last year two laboratories independently reported the discovery of a new type of stem cell that may overcome both obstacles; stem cells were found to reside in menstrual blood (Meng et al., 2007; Patel et al., 2008). These stem cells, termed endometrial regenerative cells (ERCs), are not only harvested in a noninvasive manner and relatively readily available in large quantities, but they potentially overcome the problem of immune rejection in many female patients as well.

The uterus is lined by a layer of cells called the endometrium. During the menstrual cycle, the endometrium cycles between thickening and being broken down if fertilization does not occur. The break down and expulsion of the endometrium is called menstruation, or menstrual bleeding, and is the source of endometrial regenerative cells (ERCs).

Researchers suspected stem cells to be present in menstrual blood because stem cells were previously found to be present in the lining of the uterus. The wall of the uterus is lined by a layer of cells called the endometrium (see figure). To create ideal conditions for the uterus to accept and nurture an embryo, the endometrium lining becomes thicker and increases the number of blood vessels and glands within it. However, if implantation does not occur, the endometrium lining is broken down and shed. Overall, the endometrium is quite a hyperproliferative tissue, continuously being broken down and rebuilt; it is an ideal tissue to investigate for the presence of stem cells. In the menstrual cycle, the shedding is known as menstruation, or menstrual bleeding; the excreted menstrual blood is made up of blood as well as cells from the endometrium layer. Researchers previously reported the presence of stem cells in the intact endometrium lining of the uterus (Cho et al., 2004; Schwab et al., 2005; Du and Taylor, 2007). Because stem cells were found in the endometrium, researchers thought it likely that stem cells could also be found in the shed endometrium in the form of menstrual blood, which can be obtained in relatively large quantities in a much less invasive manner. However, the stem cells discovered in menstrual blood, ERCs, appear to be rather different from stem cells derived from the intact endometrium.

While stem cells from the intact endometrium appear to be mesenchymal stem cells (MSCs, as discussed earlier), ERCs do not; they are distinctly different not only in their undifferentiated state, but in the cells they can differentiate into as well. Researchers categorize stem cells into certain groups based off of, among other factors, their cell morphology and the proteins they express. An established stem cell group usually expresses a distinct set of proteins. ERCs, though morphologically appearing mesenchymal, were found to express only some, but not all, proteins characteristic of MSCs. Additionally, ERCs were reported to be able to differentiate into, or become, cells from the three different germ layers (see the previous post on MSCs for more details): mesoderm (muscle, bone, fat, cartilage, and endothelial cells), ectoderm (neurons), and endoderm (liver, pancreas, and lung cells) (Meng et al., 2007; Patel et al., 2008). However, the mesenchymal stem cells from the intact endometrium cannot generate cells from all three germ layers. Overall, ERCs were determined to be functionally distinct from endometrium MSCs (Meng et al., 2007; Hida et al., 2008).

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admin Endometrial Regenerative Cells adult, clinical trials, embryonic, hematopoietic, mesenchymal, news, regenerative medicine

Perhaps containing more different cell types than any other stem cell category, mesenchymal stem cells (MSCs) can be isolated from a wide variety of tissues in the human body. These cells have been grouped and labeled as “mesenchymal” because they are thought to have a common progenitor in the mesenchyme, an embryonic tissue (Caplan, 2005). In the developing vertebrate embryo, there are three distinct “germ layers,” or layers of cells: the endoderm, the mesoderm, and the ectoderm. Together with the germ cells, these three layers pattern out the entire body (see figure). The mesenchyme is a collection of cells mostly derived from the mesoderm that later becomes supportive structures throughout the body, including bone, cartilage, connective tissue, smooth muscle, adipose tissue, as well as the lymphatic and hematopoietic systems. Most MSCs are thought to contain progenitors in the mesenchyme (Gilbert, 2003; Conrad et al., 2009; Caplan, 2005).

The endoderm layer later becomes skin (epidermis) and the nervous system, the ectoderm becomes the digestive tract and respiratory system, and the mesoderm becomes bone, blood, muscles, connective tissue, and several organs (heart, kidney, and gonads).

However, calling MSCs “mesenchymal” can be misleading. Because this term refers to a precursor of the large MSC family, it is referring to an embryonic tissue, though the descendant MSCs can be found in both fetal and adult tissues. MSCs have been isolated from adult muscle, bone marrow, adipose tissue, cartilage, bone, potentially teeth (Caplan, 2005) as well as some fetal tissues (fetal liver, lung, amniotic fluid, and umbilical cord) (Phinney and Prockop, 2007). The MSCs isolated from any one of these tissues are multipotent and are usually shown to be MSCs by being able to differentiate into at least three different, standard mesenchymal cell types: osteocytes (bone), chondrocytes (cartilage), and adipocytes (fat) (Baksh et al., 2004). There is much evidence, though somewhat inconsistent, showing that MSCs can also differentiate into neuronal cells, which may be from mesenchyme derived from the endoderm instead of the mesoderm (Gilbert, 2003; Phinney and Prockop, 2007). Overall, MSC differentiation potentials can vary depending on what mesenchyme-derived tissue the MSCs were harvested from (Phinney and Prockop, 2007). However, MSCs cannot become hematopoietic cells (which are derived from hematopoietic stem cells), even though these cells are derived from the mesenchyme, making the label “mesenchymal” more deceptive (Gilbert, 2003; Caplan, 2005).

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admin Mesenchymal Stem Cells adult, embryonic, hematopoietic, history, mesenchymal, regenerative medicine

Recently, a patient with leukemia and human immunodeficiency virus (HIV) had apparent remission of both after stem cell transplants (Hütter et al., 2009). As discussed earlier, hematopoietic stem cells have been used in transplants to rescue patients with leukemia, but this method has not previously been as successful for treating HIV, the virus that causes acquired immunodeficiency syndrome (AIDS).

Once in the body, HIV primarily attacks the immune system, such as T cells, though some individuals have T cells that are naturally resistant to HIV infection. Over a decade ago, this resistance was found to be due to a mutation in a receptor that is normally on the cell surface of T cells, called chemokine receptor 5 (CCR5) (Liu et al., 1996). CCR5 is a chemokine receptor, meaning it normally binds and receives signals from chemokines, which are molecules cells can release and receive to cause an immune system response. CCR5 is thought to normally be involved in causing a response to infection, though its exact function is not fully understood. HIV normally interacts with CCR5 to gain entry into the target T cell, but some individuals have a mutation in the CCR5 gene, specifically a 32 base-pair deletion, that renders the resultant receptor completely nonfunctional and consequently prevents HIV from being taken into these cells (Liu et al., 1996).

The T cell membrane (shown as the purple, semicircle double line) allows entry of HIV (in pink) into the cell through multiple cell receptors anchored on the membrane, including CCR5.

admin Hematopoietic Stem Cells adult, clinical trials, gene therapy, hematopoietic, news

Promising cures for blood-related diseases, such as leukemia and lymphoma, hematopoietic stem cells (HSCs) have been heavily researched for decades. However, like many significant findings in science, their discovery was not made in search for such a cure, but stumbled upon while dealing with another serious medical issue of the time: radiation. While trying to treat people exposed to lethal doses of radiation during World War II, transplants from the spleen and bone marrow were found to rescue these victims (Ford et al., 1956). It was not until later that scientists determined that the HSCs present in these tissues were what was restoring the damaged tissues, observed by performing transplants using lethally irradiated mouse and rat models (Becker et al., 1963). HSCs in humans were further characterized and cultured in the 1980s (Morstyn et al., 1980; Sutherland et al., 1989; Sutherland et al., 1990). The formation of the National Marrow Donor Program during this time also greatly improved the availability of these cells for research. Not only have HSCs been successfully used clinically in humans since the 1950s, but to this day they are still one of the few adult stem cells to be tested for clinical uses.

It is now not only better understood how HSCs from a donor animal can save a lethally irradiated recipient animal, but how HSCs can be used in many other medical applications as well. HSCs are able to give rise to all cells in the hematopoietic system, which includes myeloid elements (i.e. red blood cells, white blood cells, platelets) and the lymphatic system (i.e. T-Cells) (Regenerative Medicine, 2006). Because radiation generally targets rapidly dividing cells, including bone marrow cells and cells in the lymphatic system, HSCs have the ability to replenish the supply of cells most damaged by radiation. While HSCs can be collected from adult bone marrow, some fetal tissues (liver, spleen, thymus), umbilical chords, and peripheral blood, in recent years there has been a great shift towards obtaining HSCs mainly from peripheral blood, using a much simpler and less controversial procedure, though achieving a large enough number of HSCs for transplants is still an obstacle to overcome (Stem Cells, 2001). HSCs are now being used to treat cancers of the hematopoietic system (leukemia and lymphomas), replenish cells lost to high-doses of chemotherapy, and fight against autoimmune diseases, in addition to other medical applications (Regenerative Medicine, 2006).

Hematopoietic stem cells give rise to two major progenitor cell lineages, myeloid and lymphoid progenitors (Regenerative Medicine, 2006).

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