Induced stem cells

Induced stem cells (iSC) are stem cells derived from somatic, reproductive, pluripotent or other cell types by deliberate epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor (multipotent – iMSC, also called an induced multipotent progenitor cell – iMPC) or unipotent – (iUSC) according to their developmental potential and degree of dedifferentiation. Progenitors are obtained by so-called direct reprogramming or directed differentiation and are also called induced somatic stem cells.
Three techniques are widely recognized:
Natural processes.
In 1895 Thomas Morgan removed one of a frog's two blastomeres and found that amphibians are able to form whole embryos from the remaining part. This meant that the cells can change their differentiation pathway. In 1924 Spemann and Mangold demonstrated the key importance of cell–cell inductions during animal development. The reversible transformation of cells of one differentiated cell type to another is called metaplasia. This transition can be a part of the normal maturation process, or caused by an inducement.
One example is the transformation of iris cells to lens cells in the process of maturation and transformation of retinal pigment epithelium cells into the neural retina during regeneration in adult newt eyes. This process allows the body to replace cells not suitable to new conditions with more suitable new cells. In Drosophila imaginal discs, cells have to choose from a limited number of standard discrete differentiation states. The fact that transdetermination (change of the path of differentiation) often occurs for a group of cells rather than single cells shows that it is induced rather than part of maturation.
The researchers were able to identify the minimal conditions and factors that would be sufficient for starting the cascade of molecular and cellular processes to instruct pluripotent cells to organize the embryo. They showed that opposing gradients of bone morphogenetic protein (BMP) and Nodal, two transforming growth factor family members that act as morphogens, are sufficient to induce molecular and cellular mechanisms required to organize, "in vivo" or "in vitro", uncommitted cells of the zebrafish blastula animal pole into a well-developed embryo.
Some types of mature, specialized adult cells can naturally revert to stem cells. For example, "chief" cells express the stem cell marker Troy. While they normally produce digestive fluids for the stomach, they can revert into stem cells to make temporary repairs to stomach injuries, such as a cut or damage from infection. Moreover, they can make this transition even in the absence of noticeable injuries and are capable of replenishing entire gastric units, in essence serving as quiescent "reserve" stem cells. Differentiated airway epithelial cells can revert into stable and functional stem cells in vivo.
After injury, mature terminally differentiated kidney cells dedifferentiate into more primordial versions of themselves and then differentiate into the cell types needing replacement in the damaged tissue Macrophages can self-renew by local proliferation of mature differentiated cells. In newts, muscle tissue is regenerated from specialized muscle cells that dedifferentiate and forget the type of cell they had been. This capacity to regenerate does not decline with age and may be linked to their ability to make new stem cells from muscle cells on demand.
A variety of nontumorigenic stem cells display the ability to generate multiple cell types. For instance, multilineage-differentiating stress-enduring (Muse) cells are stress-tolerant adult human stem cells that can self-renew. They form characteristic cell clusters in suspension culture that express a set of genes associated with pluripotency and can differentiate into endodermal, ectodermal and mesodermal cells both in vitro and in vivo.
Other well-documented examples of transdifferentiation and their significance in development and regeneration were described in detail.
Induced totipotent cells.
SCNT-mediated.
Induced totipotent cells can be obtained by reprogramming somatic cells with somatic-cell nuclear transfer (SCNT). The process involves sucking out the nucleus of a somatic (body) cell and injecting it into an oocyte that has had its nucleus removed
Using an approach based on the protocol outlined by Tachibana et al., hESCs can be generated by SCNT using dermal fibroblasts nuclei from both a middle-aged 35-year-old male and an elderly, 75-year-old male, suggesting that age-associated changes are not necessarily an impediment to SCNT-based nuclear reprogramming of human cells. Such reprogramming of somatic cells to a pluripotent state holds huge potentials for regenerative medicine. Unfortunately, the cells generated by this technology are potentially not completely protected from the immune system of the patient (donor of nuclei), because they have the same mitochondrial DNA, as a donor of oocytes, instead of the patients mitochondrial DNA. This reduces their value as a source for autologous stem cell transplantation therapy; as for the present, it is not clear whether it can induce an immune response of the patient upon treatment.
Induced androgenetic haploid embryonic stem cells can be used instead of sperm for cloning. These cells, synchronized in M phase and injected into the oocyte, can produce viable offspring.
These developments, together with data on the possibility of unlimited oocytes from mitotically active reproductive stem cells, offer the possibility of industrial production of transgenic farm animals. Repeated recloning of viable mice through a SCNT method that includes a histone deacetylase inhibitor, trichostatin, added to the cell culture medium, show that it may be possible to reclone animals indefinitely with no visible accumulation of reprogramming or genomic errors However, research into technologies to develop sperm and egg cells from stem cells raises bioethical issues.
Such technologies may also have far-reaching clinical applications for overcoming cytoplasmic defects in human oocytes. For example, the technology could prevent inherited mitochondrial disease from passing to future generations. Mitochondrial genetic material is passed from mother to child. Mutations can cause diabetes, deafness, eye disorders, gastrointestinal disorders, heart disease, dementia, and other neurological diseases. The nucleus from one human egg has been transferred to another, including its mitochondria, creating a cell that could be regarded as having two mothers. The eggs were then fertilised and the resulting embryonic stem cells carried the swapped mitochondrial DNA.
As evidence that the technique is safe, the author of this method points to the existence of the healthy monkeys that are now more than four years old – and are the product of mitochondrial transplants across different genetic backgrounds.
In late-generation telomerase-deficient (Terc−/−) mice, SCNT-mediated reprogramming mitigates telomere dysfunction and mitochondrial defects to a greater extent than iPSC-based reprogramming.
Other cloning and totipotent transformation achievements have been described.
Obtained without SCNT.
Recently, some researchers succeeded in getting totipotent cells without the aid of SCNT. Totipotent cells were obtained using epigenetic factors such as oocyte germinal isoform of histone.
Reprogramming in vivo, by transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice, confers totipotency features. Intraperitoneal injection of such in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic (trophectodermal) markers.
The developmental potential of mouse pluripotent stem cells to yield both embryonic and extra-embryonic lineages also can be expanded by microRNA miR-34a deficiency, leading to strong induction of endogenous retroviruses MuERV-L (MERVL).
Rejuvenation to iPSCs.
iPSc were first obtained in the form of transplantable teratocarcinoma induced by grafts taken from mouse embryos. Teratocarcinoma formed from somatic cells. Genetically mosaic mice were obtained from malignant teratocarcinoma cells, confirming the cells' pluripotency. It turned out that teratocarcinoma cells are able to maintain a culture of pluripotent embryonic stem cell in an undifferentiated state, by supplying the culture medium with various factors. In the 1980s, it became clear that transplanting pluripotent/embryonic stem cells into the body of adult mammals, usually leads to the formation of teratomas, which can then turn into a malignant tumor teratocarcinoma. However, putting teratocarcinoma cells into the embryo at the blastocyst stage caused them to become incorporated in the inner cell mass and often produced a normal chimeric (i.e. composed of cells from different organisms) animal. This indicated that the cause of the teratoma is a dissonance - mutual miscommunication between young donor cells and surrounding adult cells (the recipient's so-called "niche").
In August 2006, Japanese researchers circumvented the need for an oocyte, as in SCNT. By reprograming mouse embryonic fibroblasts into pluripotent stem cells via the ectopic expression of four transcription factors, namely Oct4, Sox2, Klf4 and c-Myc, they proved that the overexpression of a small number of factors can push the cell to transition to a new stable state that is associated with changes in the activity of thousands of genes.
Reprogramming mechanisms are thus linked, rather than independent, and are centered on a small number of genes.
IPSC properties are very similar to ESCs. iPSCs have been shown to support the development of all-iPSC mice using a tetraploid (4n) embryo, the most stringent assay for developmental potential. However, some genetically normal iPSCs failed to produce all-iPSC mice because of aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster. A team headed by Hans Schöler (who discovered the Oct4 gene back in 1989) showed that Oct4 overexpression drives massive off-target gene activation during reprogramming deteriorating the quality of iPSCs. Compared to OSKM (Oct4, Sox2, Klf4 and c-Myc) that show abnormal imprinting and differentiation patterns, SKM (Sox2, Klf4 and c-Myc) reprogramming generates iPSCs with high developmental potential (nearly 20-fold higher than that of OSKM) equivalent to embryonic stem cell, as determined by their ability to generate all-iPSC mice through tetraploid embryo complementation
An important advantage of iPSC over ESC is that they can be derived from adult cells, rather than from embryos. Therefore, it becomes possible to obtain iPSC from adult and even elderly patients.
Reprogramming somatic cells into iPSCs leads to rejuvenation. It was found that reprogramming leads to telomere lengthening and subsequent shortening after their differentiation back into fibroblast-like derivatives. Thus, reprogramming leads to the restoration of embryonic telomere length, and hence increases the potential number of cell divisions otherwise limited by the Hayflick limit.
However, because of the dissonance between rejuvenated cells and the surrounding niche of the recipient's older cells, the injection of his own iPSC usually leads to an immune response, which can be used for medical purposes, or the formation of tumors such as teratoma. A hypothesized reason is that some cells differentiated from ESCs and iPSCs in vivo continue to synthesize embryonic protein isoforms. So, the immune system might detect and attack cells that are not cooperating properly.
A small molecule called MitoBloCK-6 can force the pluripotent stem cells to die by triggering apoptosis (via cytochrome c release across the mitochondrial outer membrane) in human pluripotent stem cells, but not in differentiated cells. Shortly after differentiation, daughter cells became resistant to death. When MitoBloCK-6 was introduced to differentiated cell lines, the cells remained healthy. The key to their survival was hypothesized to be due to the changes undergone by pluripotent stem cell mitochondria in the process of cell differentiation. This ability of MitoBloCK-6 to separate the pluripotent and differentiated cell lines has the potential to reduce the risk of teratomas and other problems in regenerative medicine.
In 2012, other small molecules (selective cytotoxic inhibitors of human pluripotent stem cells – hPSCs) were identified that prevented human pluripotent stem cells from forming teratomas in mice. The most potent and selective compound of them (PluriSIn #1) inhibits stearoyl-coA desaturase (the key enzyme in oleic acid biosynthesis), which finally results in apoptosis. With the help of this molecule, the undifferentiated cells can be selectively removed from culture. An efficient strategy to selectively eliminate pluripotent cells with teratoma potential is targeting pluripotent stem cell-specific antiapoptotic factor(s) (i.e., survivin or Bcl10). A single treatment with chemical survivin inhibitors (e.g., quercetin or YM155) can induce selective and complete cell death of undifferentiated hPSCs and is claimed to be sufficient to prevent teratoma formation after transplantation. However, it is unlikely that any kind of preliminary clearance is able to secure the replanting of iPSCs or ESCs. After the selective removal of pluripotent cells, they re-emerge quickly by reverting differentiated cells into stem cells, which leads to tumors. This may be due to the disorder of let-7 regulation of its target Nr6a1 (also known as Germ cell nuclear factor - GCNF), an embryonic transcriptional repressor of pluripotency genes that regulates gene expression in adult fibroblasts following micro-RNA miRNA loss.
Teratoma formation by pluripotent stem cells may be caused by low activity of PTEN enzyme, reported to promote the survival of a small population (0.1–5% of total population) of highly tumorigenic, aggressive, teratoma-initiating embryonic-like carcinoma cells during differentiation. The survival of these teratoma-initiating cells is associated with failed repression of Nanog, as well as a propensity for increased glucose and cholesterol metabolism. These teratoma-initiating cells also expressed a lower ratio of p53/p21 when compared to non-tumorigenic cells.
In connection with the above safety problems, the use of iPSCs for cell therapy is still limited. However, they can be used for a variety of other purposes, including the modeling of disease, screening (selective selection) of drugs, and toxicity testing of various drugs.
The tissue grown from iPSCs, placed in the "chimeric" embryos in the early stages of mouse development, practically do not cause an immune response (after the embryos have grown into adult mice) and are suitable for autologous transplantation
At the same time, full reprogramming of adult cells in vivo within tissues by transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs. Furthermore, partial reprogramming of cells toward pluripotency in vivo in mice demonstrates that incomplete reprogramming entails epigenetic changes (failed repression of Polycomb targets and altered DNA methylation) in cells that drive cancer development. However, a number of researchers later managed to carry out cyclical partial reprogramming "in vivo" by expression of the Yamanaka factors for a short period of time without subsequent carcinogenesis, thus partially rejuvenating and extending lifespan in progeroid mice. With "in vitro" method employing slightly longer periods of reprogramming (to substantially rejuvenate) cells temporarily lose their cell identity<ref>Olova, N., Simpson, D. J., Marioni, R. E., & Chandra, T. (2019). Partial reprogramming induces a steady decline in epigenetic age before loss of somatic identity. Aging cell, 18(1), e12877. 