To be or not to be. The stem cell case
The stem cell concept was not the result of a simple research project like many discoveries. The concept was built in years as an integrated understanding of multiple no apparently linked results. Around 1880 W. Roux presented data of experiments in which early embryos were spliced in halves which separately failed in developing a complete fetus. Then, in 1891 Eduard Driesch, in Freiburg Germany succeeded in separating the cells of the first cleavage of a sea urchin egg and growing from them identical twins (1). This result allowed Driesch to sustain the theory of the “cell pluripotency”, meaning that each cell retained the ability of developing an entire organism. Also in Germany, in 1909, Artur Pappenheim collected enough experimental information to sustain the theory of a “Blutstammzellen” (blood stem cell), origin of all types of blood cells. Much later, in 1936, Florence R. Sabin, from the Rockefeller Institute (USA), studied the development of the rabbit bone marrow proving the existence of a precursor cell population, with undefined morphology similar to lymphocytes, able to develop all blood cell lines (2). In 1958, Marcel Bessis, in France, demonstrated that the blood stem cells need the interaction with a stromal cell to differentiate in a red blood cells colony (3). In 1961, Till & McCulloch showed that the hematopoietic stem cells are sensitive to radiation (4). These results consolidated the technical feasibility of stem cell elimination and substitution, foundation of the bone marrow transplant, and probably the first application of regenerative medicine initiated by E.D. Thomas.
But the self renewal nature of the hematopoietic stem cell, although it was suspected, was not experimentally proven until 1968 (5). Surprisingly, from 1900 until 1970 only 129 papers are recorded in PubMed archives including the linked two words “stem cell”, mainly oriented to hematopoiesis. Along this period emerged the concept of the existence of “Stem Cells” in adult tissues, like bone marrow, capable of developing many different branches or lineages of cells (differentiated cells). Those stem cells were capable of auto-renewal following a stochastic process (6). Adding all different concepts together the attribute of “stem” was reserved for cells capable of developing “branches” by differentiation like the paradigmatic bone marrow stem cell. Because a fetus has a broad multiplicity of cells all derived from the egg this is by definition the first Stem Cell or even more, the “all potential” cell (Toti-Potent Cell) and the first embryonic cells, because they have multiple or plural potential they are considered “Pluri-Potent Cell”
Bone marrow and hematopoiesis were excellent targets for tissue regeneration research, but another tissue, the liver, was also appealing and eye-catching for multiple reasons. Nobody knows how the ancient Greeks figured out that the liver could regenerate after heavy tissue losses and had spread the myth of Prometheus, who was punished by the gods by being attacked by an eagle every day. The eagle took from Prometeus part of his liver every day, which regenerated overnight.
Since the beginning of the 20th century liver regeneration was a well known fact mainly studied in rodents. Liver is an endoderm derived tissue composed by several cell types with two major components: Hepatocytes and bile duct cells. Studies on liver tumors development suggested that both cell types derive from a single stem cell and this was identified as the “Oval Cell”, visible fundamentally in the liver of rodents, apparently located in the structure of the Hering channel (7). As soon as 1947 it was observed that liver regeneration was enhanced in hypoxia conditions (8) providing one of the first clues that the amount of oxygen has a cell regulation role in tissue regeneration (9).
However, these cells were considered more in the class of the “Reserve Cells” which the classic histologists use to find in the skin epithelium (10), in the intestinal cryptal epithelium (11), in the uterine mucosa (12), striated muscle (satellite cells) (13) and others that today are also called stem cells. Reserve cells are capable of cell renewing and able to develop or differentiate in a limited variety of local cells, but they are not pluripotent, they are already “committed" to a certain cell type differentiation, therefore if they are considered or called stem cells, these are a limited type of stem cell, that may have the ability of limited asymmetric division period followed by a symmetrical division life that produces a progeny of differentiated cells (Fig 1). It was deemed that asymmetric cell division is a crucial characteristic of stem cells enabling them to perpetuate themselves (self-renew) and generate differentiated progeny (14); however, asymmetric cell division may be also a resource that committed cells can use to maintain suitable numbers of progeny, to preserve the volume and function of regenerated tissues (15).
The unanswered questions about the pluripotent cells, and these limited-potent cells, was if they contained all genes involved in the development of a complete being or differentiation involves gene losses. In 1958, J.B. Gurdon provide an experimental solid answer by generating a frog from an un-nucleated ovule in which he transplanted the nucleus of a well differentiated intestinal epithelial cell (16), therefore the nucleus of a differentiated cell contains all genes needed for the development of a complete being (17). In addition, this experiment demonstrated that the egg cytoplasm contains factors than can “reprogram” the genetic expression of the differentiated nucleus up to a pluripotent cell nucleus. In 1988, Henry Harris summarized his research on heterokaryons demonstrating that even the tumor cells can be reprogrammed when in the cytoplasm exist certain “suppressor genes” (18). A concise research opened another line of thinking: The differentiation involves the “sequential expression” of different genes (19). These concept open the theoretical possibility of reprogramming by “altering the sequence” of the gene expression.
The beginning of an effective cell therapy started with the bone marrow transplant primarily developed by ED Thomas (20) in 1959. This valuable medical application invited the extended research in the concept and use of the stem cells (note that from 1970 to 1998 there are recorded in the PubMed archives about 3,000 publications involving stem cell matters).
Human pluripotent cell lines derived from blastocyst were first described and published in 1998 by J. Thomson et al. The authors describe them as cells with normal karyotypes, expressing high levels of telomerase activity, cell surface markers that characterize embryonic stem cells and markers which are lost in other cells of early lineages (21). Moreover, these cells can proliferate in vitro, maintaining the developmental potential to form derivatives of all three embryonic germ lines (22), meaning that they can divide symmetrically for quite a broad expansion.
Molecular biology studies identified active molecules with different regulatory functions that identified pluripotency and those become the basic instrument to identify stem cells, pluripotency traits and differentiation. Between all of them a few are the really consistent (Oct4, Klf4, Sox2, NANOG and cMyc).
In 2006 Takahashi K, Yamanaka S. demonstrate that adult fibroblasts can be reprogrammed into embryonic stem cell phenotype by introducing in them a set of specific factors ( Oct3/4, Sox2, c-Myc, and Klf4). There are the induced pluripotent stem cells (iPSC) (23). However, the iPS cells do differ from the embryonic stem cells ESC in the gene expression and DNA methylation patterns, but the additional expression of NANOG transcription factor results in germline-competent iPS cells with ES-cell-like gene expression and DNA methylation patterns (24). Thus, iPS cells competent for generation of germline chimaeras can be obtained from differentiated cells such as fibroblasts, with today established tools like OKSM which consists of four adenoviruses expressing one of four mouse transcription factors (Oct4, Klf4, Sox2 or cMyc) under the control of a promoter. However, retroviral introduction of c-Myc is dangerous for clinical application (25) cutting short the possible clinical application. Also it is possible to reprogram amniotic fluid stem cell, even more easily than adult fibroblast using ectopic expression of OKSM. Nevertheless, the iPS cells are a great promise and represent an extraordinary progress in the research and knowledge of the cell differentiation and tissue regeneration.
Cancer is a major research and practice field where stem cells are a mystery. Many people are thinking that like any other tissue the neoplastic tissues may have reserve cells capable of self renewal. It has been for a long time admitted that malignant tumors shed cells that are transported to other sites to reproduce the same neoplastic tissue in different organs (metastases). From this point of view there is no doubt that those tumors have at least “reserve cells”, better called “metastatic cells” that have a specific phenotype and can reproduce the tumor from one or a few cells. For more than 40 years, it has been well known that cancer and metastatic tumor cells can show expression of fetal/embryonic genes (26)(27) as a sign of retro differentiation (28). Recently the “stem cells” are fashionable and there are in the journals a plethora of papers (29) claiming the finding of cells that are called “stem cells” because, as shown 30 years ago, they are cells that express one or more retro differentiation genes. Many prudent people call these tumor colony forming cells, “cancer stem-like cells” (CSCs) or “tumor-initiating cells”, which is closer to how they have been considered for years. However, to be “stem” is not only a matter of showing active embryonic genes. To be STEM means that the cell is capable of both self-renewal and differentiation. A metastatic cell is capable of self renewal, of course, but it is not clear if it is capable of developing stable branches or lineages of cells with specific functions and traits. Many scientists claim that tumors are formed by a variety of different cells with different traits and those are enough to confer the honor of STEM to the tumor initiating cells (30). On the other hand, it is not yet clear if each group of cells of this heterogeneous population can be considered a branch or a linage with consistent differentiation (31). The evidences are in better agreement with very unstable clonal variations due to division errors, DNA damage, replication errors, immune editing and responses to different stressing environments (32). A cell lineage or a branch of stem cells requires a stochastic process of setting on and off a collection of genes under a program existing in the egg cell. For it is known of the oncogenesis, it is not clear that cancer cells are following a well defined program of differentiation, on the contrary it looks like they have lost the control of the genetic program inherited from the normal stem cell or the tissue reserve cells.
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