Genetic and Epigenetic Instability in Human Induced Pluripotent Stem Cells (Essay Sample)

GENETIC AND EPIGENETIC INSTABILITY IN HUMAN INDUCED PLURIPOTENT STEM CELLS (hiPSC)
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Genetic and Epigenetic Instability in Human Induced Pluripotent Stem Cells (hiPSC)
Genetics and epigenetics variation and instabilities have been reported by several high-resolution genomic studies in the recent past irregularly to accumulate in human pluripotent stem cells. Detailed characterization of these changes in the proliferation, self-renewal and mainly on the developmental and malignant potential of these cells is essential for the understanding alteration the impact. The characterization is important for the enhanced and safe use of pluripotent cells for therapeutic use, such as regenerative cellular therapies utilizing differentiated derivatives of pluripotent cells. In this review, epigenetic and genomic stability of human pluripotent cells and the implications for stem cell research will be summarized.
An extensive study on genome stability for the human pluripotent stem cells and their derivatives, utilizing a combination of high-resolution genomic methods has been carried out. The structural genomic aberrations have been carried out by combining multiplex fluorescence in an in situ hybridization (M-FISH) and array comparative genomic hybridization (aCGH) techniques. Fascinatingly, different chromosomal and subchromosomal genomics have been identified in separate research and described amongst pluripotent stem cells and their derivatives. Some of the acquired modifications in the copy number variation are presumably acquired during the differentiation process and manipulation procedures while some of the other variations observed are probably inherited from the original cell lines. The need to study both the pluripotent stem cells and their differentiated stem cells using various methods arose from the previous findings, so as to evaluate and a certain their stability before using them in clinical therapies.
The necessity to develop therapies has increased the improvements in the differentiation and culture methods, legal permissions, industrial-scale cell culture and considerable investment from both private and public entities. However, as the concerns about the safety of hPSC therapies remain a major obstacle the production continuous. In other incidences, the transformed cells may harm the patient by inducing cancer or becoming cancerous. Two major concerns about tumorigenicity arise when it comes to cell types that are derived from human pluripotent stem cells (hPSCs)( Gore et al., 2011).Firstly, mutations in transplanted cells are a concern; genomic mutations are associated with tumorigenicity development. Secondly, undifferentiated human pluripotent stem cells can give rise to germ cell tumors that are normally benign when transplanted into mice that are immune-deficient during various researches. Though there are various concerns about residual undifferentiated cells in transplanted (hPSC) populations because all the clinical applications that are carried out require that the cells to be differentiated first before the process can commence. A more thorough characterization of the pluripotent stem cells and their derivatives of neuronal progenitor cell lines research first described structural genomic alterations in the primate’s stem cells (Thomson and Marshall, 1998). Since then the various research on genomic anomalies that have preceded the research by Thomson and Marshall (1998) have been identified in almost every single type of pluripotent stem cells, in spite of the cell culture condition used, modifications or manipulations incurred during the process. By comparison, epigenetic modifications are sensitive and dynamic to environmental stress and signals as was described by Robert Feil & Mario F. Fraga (2012) in their review journal. However, genomic variations and instabilities lack enough information that could potentially be got from differentiated acquired pluripotent stem cell types.
If acquired pluripotent cell types would be used as model in basic research or cell therapy, it’s essential that their genomic integrity should be thoroughly characterized and analyzed extensively so as to avoid any potential danger that they might pose to a patient. The idea that tumorigenicity and cancerous variants could grow in a relatively shorter duration was dismissed after covering the completion of a typical differentiation procedure that took 1 to 3 weeks (Stephenson et al, 2010; Winkler et al, 2009). According to a separate research conducted by Tomazou et al., (2010) all human pluripotent stem cells and human embryonic stem cells hold a very big potential for clinical and therapeutic applications. This is because they hold differentiation capacities and very incredible self-renewal they hold that makes them a possible source for large-scale quantities of differentiated cells for therapeutic biomolecule production, drug screening, cell therapy and toxicological studies and experiments.
In a highly replicated study for the identification of passing effects that might rise from various methods used and differential substrate constituents taking longer time in culture on epigenetic and genetic instabilities and phenotypic characterization of human pluripotent stem cells was achieved by Biancotti et al (2010). In both the research on human-induced pluripotent stem cells and human pluripotent stem cell researches. They observed that various accumulation in genetic changes and enzymatic passaging on a feeder free substrate was directly proportional to the changes that are seen in mechanical passaging on feeder layers. They observed in their study that extensive culture of human embryonic stem cells over a long duration of time under the conditions mentioned above indicated that even when used in conditions with the lowest overall rate of genetic alterations the number of changes rose drastically after going through 80 passages (which breaks down to a total number of 80 + 37=117). Another more detailed study on human embryonic stem cells indicated that the enzymatic passaging had minimal, or no effects at all on the buildup of genetic substrates compared to aberrations (Winkler et al., 2009).
Various methods for creating pluripotent stem cells by reprogramming for somatic cells from other tissues and species by the ectopic expression of distinct factors
Application

Species of choice

Donor cell type

Reprogramming cocktail

Delivery mode

Recommendations

Drug screening and disease modeling.

Human/pig

Reprogrammable cells easily available from patients or cell repository

OSK/OSKM; OSNL

Retrovirus; RNA?

The starting cell population may be limited, so competent methods are needed to generate models. Safety is not an important issue but avoiding integration would decrease genetic heterogeneity among cell lines

Study reprogramming mechanism

Mouse

Cells from chimeric mice from iPSCs obtained using an inducible system.

OSK/OSKM* as reference, any additional factor possible

Inducible lentivirus

To understand reprogramming, by comparison of as many factors and cell types as possible

Cell therapy

Human

Reprogrammable cells readily available from cell repository, patients, or HLA-matched iPSCs obtained from cord blood

Need to avoid inhibitors of tumor suppressor or potent oncogenes.

Non-integrative

Non-integrative

Research on pluripotency/differentiation

Human/ Mouse

mouse embryonic fibroblasts (MEFs)/fibroblasts

*OSKM/OSK; OSNL‡

Retrovirus; RNA?

To improve differentiation protocols and understand pluripotency, reproducible and reliable reprogramming methods are amongst best. Non-integrative methods may reduce genetic heterogeneity among cell lines

Subject to specific purpose of reprogramming, many methods have been developed when considering how to generate induced pluripotent stem cells (iPSCs); human leukocyte antigen (HLA); mouse embryonic fibroblasts (MEFs).
Shinya Yamanaka developed the * OSKM and *OSK combinations. In which *OSK demonstrates the combination of various transcription factors (TFs) SOX2, OCT4 and KLF4, and OSKM describes the combination of KLF4, OCT4, SOX2 and MYC.
James Thomson developed the ‡ OSNL combination. In which OSNL describes the combination transcription factors (TFs) OCT4, NANOG, LIN28 and SOX2, NANOG also known as Thomson factors.
Genetic instability
Karyotypic abnormalities such as trisomy of the chromosome had begun emerging in human embryonic stem cells begun emerging in early 2000. Some of this abnormities were reported by Hardarson et al. (2003). Later on other abnormalities were discovered by Laurent et al (2013) in their research; sub-chromosomal aberrations to whole chromosome aneuploidy, including point mutation are just some genome abnormalities that were indicated in their findings on their researched in human pluripotent stem cells studies. These findings were also supported with independent research carried out by Woltjen et al. (2009). Some duplication were also observed to be recurrent in human pluripotent stem cell studies (Woltjen et al. 2009)
Does human induce pluripotent stem cell reprogramming influence genetic stability?
According to Takahashi et al. (2007) in their research the first-generation of human induce pluripotent stem cell (hiPSC) lines were reprogrammed from somatic cells using lentiviral and retroviral vectors, which randomly integrate into the genome of the parent cell. Several other footprint free protocols have been developed such as those hypotheses by several separate research (Kim et al. 2009; Woltjen et al.,2009; Waren et al., 2011; Miyoshi et al.,2011) to minimize the risk of harmful re-activati...