Lineage-instructive approach

In this approach, transcription factors from progenitor cells of the target cell type are transfected into a somatic cell to induce transdifferentiation.^[2] There exists two different means of determining which transcription factors to use: by starting with a large pool and narrowing down factors one by one^[18] or by starting with one or two and adding more.^[19] One theory to explain the exact specifics is that ectopic Transcriptional factors direct the cell to an earlier progenitor state and then redirects it towards a new cell type. Rearrangement of the chromatin structure via DNA methylation or histone modification may play a role as well.^[20] Here is a list of in vitro examples and in vivo examples. In vivo methods of transfecting specific mouse cells utilize the same kinds of vectors as in vitro experiments, except that the vector is injected into a specific organ. Zhou et al. (2008) injected Ngn3, Pdx1 and Mafa into the dorsal splenic lobe (pancreas) of mice to reprogram pancreatic exocrine cells into β-cells in order to ameliorate hyperglycaemia.^[21]

Initial epigenetic activation phase approach

Somatic cells are first transfected with pluripotent reprogramming factors temporarily (Oct4, Sox2, Nanog, etc.) before being transfected with the desired inhibitory or activating factors.^[22] Here is a list of examples in vitro.

Pharmacological agents

The DNA methylation inhibitor, 5-azacytidine is also known to promote phenotypic transdifferentiation of cardiac cells to skeletal myoblasts.^[23]

In prostate cancer, treatment with androgen receptor targeted therapies induces neuroendocrine transdifferentiation in a subset of patients.^[24][25] No standard of care exists for these patients, and those diagnosed with treatment induced neuroendocrine carcinoma are typically treated palliatively.^[26]

Mechanism of action

The transcription factors serve as a short term trigger to an irreversible process. The transdifferentiation liver cells observed 8 months after one single injection of pdx1.^[12]

The ectopic transcription factors turn off the host repertoire of gene expression in each of the cells. However, the alternate desired repertoire is being turned on only in a subpopulation of predisposed cells.^[27] Despite the massive dedifferentiation – lineage tracing approach indeed demonstrates that transdifferentiation originates in adult cells.^[28]

Mogrify algorithm

Determining the unique set of cellular factors that is needed to be manipulated for each cell conversion is a long and costly process that involved much trial and error. As a result, this first step of identifying the key set of cellular factors for cell conversion is the major obstacle researchers face in the field of cell reprogramming. An international team of researchers have developed an algorithm, called Mogrify(1), that can predict the optimal set of cellular factors required to convert one human cell type to another. When tested, Mogrify was able to accurately predict the set of cellular factors required for previously published cell conversions correctly. To further validate Mogrify's predictive ability, the team conducted two novel cell conversions in the laboratory using human cells, and these were successful in both attempts solely using the predictions of Mogrify.^[29][30][31] Mogrify has been made available online for other researchers and scientists.

Evaluation

When examining transdifferentiated cells, it is important to look for markers of the target cell type and the absence of donor cell markers which can be accomplished using green fluorescent protein or immunodetection. It is also important to examine the cell function, epigenome, transcriptome, and proteome profiles. Cells can also be evaluated based upon their ability to integrate into the corresponding tissue in vivo^[18] and functionally replace its natural counterpart. In one study, transdifferentiating tail-tip fibroblasts into hepatocyte-like cells using transcription factors Gata4, Hnf1α and Foxa3, and inactivation of p19(Arf) restored hepatocyte-like liver functions in only half of the mice using survival as a means of evaluation.^[32]

Transition from mouse to human cells

Generally transdifferentiation that occurs in mouse cells does not translate in effectiveness or speediness in human cells. Pang et al. found that while transcription factors Ascl1, Brn2 and Myt1l turned mouse cells into mature neurons, the same set of factors only turned human cells into immature neurons. However, the addition of NeuroD1 was able to increase efficiency and help cells reach maturity.^[33]

Order of transcription factor expression

The order of expression of transcription factors can direct the fate of the cell. Iwasaki et al. (2006) showed that in hematopoietic lineages, the expression timing of Gata-2 and (C/EBPalpha) can change whether or not a lymphoid-committed progenitors can differentiate into granulocyte/monocyte progenitor, eosinophil, basophil or bipotent basophil/mast cell progenitor lineages.^[34]

Immunogenicity

It has been found for induced pluripotent stem cells that when injected into mice, the immune system of the synergeic mouse rejected the teratomas forming. Part of this may be because the immune system recognized epigenetic markers of specific sequences of the injected cells. However, when embryonic stem cells were injected, the immune response was much lower. Whether or not this will occur within transdifferentiated cells remains to be researched.^[3]

Method of transfection

In order to accomplish transfection, one may use integrating viral vectors such as lentiviruses or retroviruses, non-integrating vectors such as Sendai viruses or adenoviruses, microRNAs and a variety of other methods including using proteins and plasmids;^[35] one example is the non-viral delivery of transcription factor-encoding plasmids with a polymeric carrier to elicit neuronal transdifferentiation of fibroblasts.^[36] When foreign molecules enter cells, one must take into account the possible drawbacks and potential to cause tumorous growth. Integrating viral vectors have the chance to cause mutations when inserted into the genome. One method of going around this is to excise the viral vector once reprogramming has occurred, an example being Cre-Lox recombination^[37] Non-integrating vectors have other issues concerning efficiency of reprogramming and also the removal of the vector.^[38] Other methods are relatively new fields and much remains to be discovered.