Direct Reprogramming: Turning One Cell Directly Into Another
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.
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).
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.
The common myeloid progenitors can directly become megakaryocytes or erythrocytes. (Megakaryocytes reside in the bone marrow and generate platelets (thrombocytes), which are necessary for blood clotting. Erythrocytes, or red blood cells, are the most common blood cell and deliver oxygen to the body through the blood system.) Common myeloid progenitors can also become monocytes and eosinophils, but to do this it must first become a myeloblast. (Monocytes are white blood cells that create macrophages, while eosinophils are white blood cells that combat infections.)
With this understanding of hematopoiesis, the importance of the 1995 report (Kulessa et al., 1995) becomes clearer. Their findings showed that when high levels of GATA-1 were expressed in monocyte precursors (cells that have not yet fully differentiated into monocytes), these cells could dedifferentiate into cells that occurred an earlier point in hematopoiesis differentiation, the erythrocytes and megakaryocytes. This makes sense with GATA-1’s normal role in hematopoiesis; GATA-1 is an important transcription factor for erythrocyte and megakaryocyte differentiation. GATA-1 is expressed in hematopoietic progenitors, but becomes downregulated in monocytes during differentiation. Interestingly, when lower levels of GATA-1 were expressed, the monocytes became eosinophils; these lower levels are normally present in eosinophils (Kulessa et al., 1995).
While all of the cells in this study were descendants of common myeloid progenitors, it was shown in 2004 that descendants of the other hematopoietic branch, those derived from common lymphoid progenitors, could also be coaxed into becoming a descendant of common myeloid progenitors (Xie et al., 2004). Common lymphoid progenitors can normally become B-cells, also called B lymphocytes (white blood cells that make antibodies against invaders). In 2004, it was reported that B-cells could be reprogrammed into macrophages by making the B-cells express C/EBP transcription factors (C/EBP stands for CCAAT-enhancer-binding proteins). C/EBPs are necessary for cells to normally differentiate from monocytes into macrophages. Interestingly, B-cell progenitors much more efficiently became macrophages than fully differentiated B-cells, again emphasizing the key role that the differentiation state plays in the ability to reprogram a cell. This 2004 report was also significant in that it was the first report showing that fully differentiated cells could be reprogrammed using transcription factors; the first report of transdifferentiation.
While C/EBPs work to enforce a macrophage fate, they also actively work to prevent the B-cell fate. C/EBPs inhibit Pax5 (paired box gene 5), which is a transcription factor that reinforces the B-cell’s commitment (Nutt et al., 1999; Xie et al., 2004). The function of Pax5 has been investigated through ablation studies; when the Pax5 gene is deleted, B-cells become dedifferentiated, turning into common lymphoid progenitor-like cells, which can then be differentiated into T-cells (lymphocytes) (Cobaleda et al., 2007). However, this is not quite direct reprogramming, as it requires the lymphoid progenitor cell state. (T-cells can also be reprogrammed using C/EBPs; its expression can induce T-cells to undergo macrophage differentiation (Laiosa et al., 2006).)
Most recently, reprogramming in the hematopoietic system also taught researchers an important reprogramming lesson: the order in which the cells are exposed to the transcription factors affects reprogramming, probably in a way similar to in vivo (Graf and Enver, 2009).
While the hematopoietic system appears to have some rather flexible cells differentiation-wise, it was some time before such reprogramming abilities were proven in other cellular systems. In 2008, the ability to reprogram one type of pancreatic cell, exocrine cells, into a functionally different type, beta-cells, was reported (Zhou et al., 2008). Exocrine cells are highly specialized pancreatic cells which produce digestive enzymes for the small intestine. Beta-cells (http://en.wikipedia.org/wiki/Beta_cell) reside in the islets of Langerhans, inside the pancreas, where they produce insulin, a hormone that regulates blood glucose levels. Insulin stimulates multiple organs to take glucose in their cells from the blood stream. Diabetes can develop due to high blood glucose levels, caused by the body not producing enough insulin or not responding to insulin it produces. Because diabetes can be caused by a lack of insulin production, the ability to create beta-cells is quite appealing.
From the start, the group set out to find the key transcription factors that could reprogram exocrine cells into beta-cells. They screened over 1,100 transcription factors and found around 20 were only expressed in mature beta cells, and 9 of these caused an abnormal developmental phenotype when mutated, indicating their functional importance in the development of the pancreas. These 9 were used for the initial reprogramming screens in mice, using adenoviral vectors to infect only the pancreatic exocrine cells. The studies were done in mice, and not in culture, to let the natural environment aid in survival and maturation of the cells and allow for direct comparisons of the reprogrammed cells to the native beta-cells. Ultimately, the combination of transcription factors that worked best was Ngn3 (Neurogenin3), Pdx1, and Mafa. Expressing these factors resulted in exocrine cells becoming beta-like-cells that had the same size, shape, structure, and protein expression as native beta-cells, and could also produce insulin (Zhou et al., 2008). However, while exocrine cells and beta cells are functionally quite different, both are derived from the pancreatic endoderm; it still remained to be seen whether more developmentally removed cells could be reprogrammed into each other.
Fibroblasts and Neurons
The most recent breakthrough on direct reprogramming of cells reported the ability to convert fibroblasts into neurons (Vierbuchen et al., 2010). Specifically, the researchers used mouse embryonic fibroblasts and postnatal fibroblasts and, using three transcription factors known to be important in specifying the neural-lineage fates, made the cells into functional neurons in vitro. The researchers first tested 19 candidate transcription factors, chosen for their expression in neural cells or their ability to reprogram cells to pluripotency. Infecting the fibroblasts using lentiviral vectors, the researchers screened for the ability of the candidates to induce a neuronal phenotype, and indeed found some that became neuronal-like. The researchers narrowed down the candidates to a smaller group to see what was necessary for the neuronal-like phenotype, and discovered three transcription factors to be key: Ascl1, Brn2, and Myt1l. While Ascl1 alone could induce immature neuronal features, the other two were required for mature neuron-like cells. The resultant neurons expressed neuron-specific proteins and functioned like neurons (they could generate action potentials and form functional synapses).
While direct reprogramming of adult cells into other cell types is clearly possible, the process by which it happens remains largely not understood. Much research needs to be done to understand the vital molecular mechanisms at play, as well as what occurs at the cellular level. Specifically, it is unclear whether, during direct reprogramming experiments, a cell turns into a progenitor briefly and then differentiates into the final cell type, or the cell actually differentiates directly to the final cell type (Graf and Enver, 2009).
Direct reprogramming efforts in the future may incorporate many factors in addition to transcription factors to be most effective. Studies are already testing the effects of altering expression of microRNAs and factors involved in chromatin remodeling, along with effective chemicals, on cell identity and differentiation; in the future, these approaches will most likely be used along with changing the expression of key transcription factors to find the most effective combinations (Graf and Enver, 2009). Additionally, many studies to date have been in mice and mouse cells; these must be repeated with human cells before they can be used clinically in humans. The resultant cells will be important not only for creating patient-specific cells for cellular therapies and regenerative medicine, but also for studying cell differentiation, plasticity during development, and cell identity problems that occur during diseases such as cancer.
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