The Possibilities Of Direct Transdifferentiation Biology Essay

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For the last couple of decades stem cells have been a hot topic within the sciences of biomedical research. Human embryonic stem (ES) cells derived from the inner cell mass of the mammalian blastocyst can grow indefinitely while maintaining pluripotency, which is defined by the ability to differentiate into all tissues of the body. (ref)

However, ethical and political issues and problems with immunological rejection of ES raised the need of alternative methods and in 2006 the first results of induced pluripotent stem cells were published. Though a great contester there are still some hurdles that need to be overcome and the possibilities of direct transdifferentiation might be the solution.

A variety of applications have been proposed for pluripotent stem cells, including disease modeling, screens for drug discovery, and tissue engineering for degenerative diseases.

The aim of this review is to answer the question: Is it possible to perform transdifferentiation of fibroblasts into fully functional cells of a different lineage?

To answer this broad question several aspects are discussed. Methods will be compared, the possible differences between de iPS techniques and the way transdifferentiation is performed. Furthermore this review will discuss the controls that have been performed by the research groups, are the resulting induced cells identical to the intended normal cells? Finally, the possibilities and restrictions of the clinical use of transdifferentiated cells within the field of regenerative medicine is discussed.

Embryonic and adult stem cells

Stem cells are characterized by being undifferentiated and having the ability to generate new stem cells, but can also be induced to become specialized cell types by certain physiological and experimental conditions. {{168 Evans,M.J. 1981; 167 Thomson,J.A. 1998}} A stem cell has the ability to divide into another stem cell a process also called self-renewal or it can divide into a cell that has the potential to differentiate into another type of cell with a specialized function, such as a neuron , blood cell or muscle cell.

Stem cells can be classified into four categories, respectively totipotent, pluripotent, multipotent and unipotent. Totipotent stem cells have the potential to differentiate into cell types of all the tissues present in the human body, including the embryonic membranes and placenta. Pluripotent stem cells are present in the inner cell mass of a blastocyst. They can differentiate into cell types of all the three germ layers; ectoderm, mesoderm and endoderm. Multipotent stem cells are able to differentiate into a smaller spectrum of cells restricted to one of the three germ layers. Finally as the name suggests unipotent stem cells are cells that can differentiate into only one cell type.{{167 Thomson,J.A. 1998}}

Embryonic stem cells are able to generate cell types of clinical interest with relatively much ease and this quickly led to much enthusiasm, however the adult stem cells are responsible for maintaining tissue homeostasis by replacing cells that have been lost due to maturation, aging or damage. For this reason, they also hold great promise for tissue regeneration and repair. Furthermore, stem cells have been become increasingly useful for fundamental research since they can be expanded in vitro while maintaining their native properties. For example, scientists have been able to create cardiomyocytes {{172 Xu,C. 2002}}, neurons {{173 Carpenter,M.K. 2001; 174 Bjorklund,L.M. 2002}} and beta cells {{175 Assady,S. 2001}} using embryonic stem cells. Consequently, speci¬c stem cells have become a reference model for understanding key molecular mechanisms that control cell fate and tissue differentiation.

But stem cells are not a thing of just the laboratory anymore, in fact haematopoietic stem cells have been successfully used in the clinic for the last 40 years for treatment of diverse blood disorders as leukaemia. {{171 Burt,R.K. 2008}}

Recent developments

Induced Pluripotent Stem cells

Increased research and use of embryonic stem cells created its own need for an alternative. The political and ethical implications about the use of embryos had been controversial from the start and a non-embryonic pluripotent cell was sought after.

In 2006 the first results were published that showed the potential of replacing just a few genes to induce pluripotency of adult mouse fibroblasts.(Takahashi & Yamanaka, 2006) Only one year later, the same group were able to do the same with human orthologues and human fibroblasts.(Takahashi et al., 2007) Now, several groups have created recipes and different methods of activating specific genes to create these induced pluripotent stem cells (iPSC). Masip et al have documented and organized the current research on iPS cells and provide a well structured table that includes information of the cell source, the reprogramming factors used and the reprogramming efficiency.(Masip, Veiga, Izpisua Belmonte, & Simon, 2010) This article will continue on the methods used and possible differences between studies not limited to the use of different reprogramming factors.

How do iPS cells come about?

As mentioned earlier, just a few genes are sufficient to induce pluripotency. These genes, referred to as the transcription factor octet consists of Oct4, Sox2, Klf4 and c-Myc.

Low efficiency!

Not only are iPS useful in regenerative medicine, diseased cells are also being used as a template for pathology research and therapeutic strategies. This disease modelling is already done for research of diseases such as adenosine deaminase deficiency-related severe combined immunodeficiency, Scwachman-Bodian-Diamond syndrome, Gaucher disease type III, Down Syndrome, Huntington disease, Parkinson disease, Duchenne and Becker muscular dystrophy, type 1 diabetes mellitus and Lesch-Nyhan syndrome (carrier state). (Park et al., 2008)

iPS en Regenerative medicine..

Direct Transdifferentiation

A primary goal of regenerative medicine is to produce new cells to repair or replace diseased and damaged tissues. One interesting and promising idea is that of changing existing adult cells into new ones by converting them from one cell type to another. Abundant human cells such as dermal ¬broblasts and adipocytes could be harvested and converted into other, medically important cells such as neurons, cardiomyocytes, blood cells or pancreatic β-cells. (figuur)

Figure 1 Overview of the described studies. Fibroblasts have been used to convert to many cells of different lineages. The genes used for conversion are listed in the figure. iPS; induced pluripotent stem cell

Fibroblasts to muscle using MyoD

From fibroblast to macrophage

It's not until early 2008 that more reports emerge on the topic of transdifferentiation.

The report of Feng et al. describes how they were able to convert fibroblasts into macrophage-like cells. The combination of PU.1 and C/EBPα, as well as PU.1 and C/EBPβ , induced the upregulation of macrophage/hematopoietic cell surface markers in a large proportion of NIH 3T3 cells. They also up-regulated the macrophage markers in mouse embryo- and adult skin-derived ¬broblasts. Approximately 70% of the embryo-derived fibroblasts coinfected with PU.1 and C/EBPα expressed Mac-1, a myelomonocytic marker, with adult-derived cells showing percentages approximately two times lower.Based on cell morphology, activation of macrophage-associated genes, and extinction of ¬broblast-associated genes, cell lines containing an attenuated form of PU.1 and C/EBP acquired a macrophage-like phenotype.

However (bleven niet macrofagen, expressie naar beneden. Wellicht nog andere stabilizerende factoren nodig)


Exocrine cells to B-cells

Another report from 2008 was that of the research group of Qiao Zhou who reported that they identified a specific combination of three transcription factors (Ngn3 (also known as Neurog3) Pdx1 and Mafa) that reprograms differentiated pancreatic exocrine cells in adult mice into cells that closely resemble b-cells. They carried out the experiments in vivo so that any induced b-cells would reside in their native environment, the authors reasoned that this might promote the survival and maturation of the cells. The transcription factors were delivered into the pancreas in adenoviral vectors.

More than 20% of the infected cells were converted to insulin positive cells, however the reprogramming effect of the three factors appeared to be rather specific for pancreatic exocrine cells: infection of skeletal muscle in vivo or fibroblasts in vitro with M3 did not induce insulin expression. (Zhou, Brown, Kanarek, Rajagopal, & Melton, 2008)

Fibroblasts to blood cells

Szabo et al. were able to transform human dermal fibroblasts into cells of the haematopoietic lineage bypassing a pluripotent state. By ectopic expression of OCT4 and using a cytokine treatment (SCF, G-CSF, FLT3LG, IL-3, IL-6 and BMP-4) they were able to generate cells expressing the pan-hematopoietic cell marker CD45. It was not until this study that OCT4 was implicated in blood cell development and other iPS reprogramming factors did not have this effect.

The authors studied the expression of genes associated with pluripotency and demonstrated that the resulting induced cells did not enter a pluripotent state. They were also able to show that the induced cells were able to mature into myeloid lineages in vitro and able to engraft in a mouse in vivo.

(Szabo et al., 2010)

From fibroblasts to cardiomyocytes

Another study done by Ieda et al reported that a combination of the developmental factors Gata4, Mef2c, and Tbx5 reprogrammed postnatal cardiac or dermal ¬broblasts directly into differentiated cardiomyocyte-like cells.

To ensure that they were not measuring cardiomyocytes from the start they screened the fibroblast cells on the expression of GFP and the cardiac fibroblast markers Thy1 and vimentin. Mature cardiomyocytes would express GFP while the fibroblasts would be GFP negative. Cells were isolated by FACS and confirmed that they had no cells expressing the cardiomyocyte marker troponin T.

They determined 14 factors critical for cardiac gene expression but by removing individual factors they ended up with 3 that increased the number of fibroblasts that expressed GFP dramatically. When removing one of these 3 factors neither GFP expression nor troponin T markers were detected.

The expression profile of the induced cardiomyocytes was tested and was found similar to that of neonatal cardiomyocytes which probably accounts for the fact that the induced cells possess similar functional properties to that of neonatal cardiomyocytes. For example, they found spontaneous Ca2+ oscillations in 30% of the induced cells. Furthermore the intracellular electrical recordings of the induced cells resembled those of adult mouse ventricular cardiomyocytes.(Ieda et al., 2010)

Fibroblasts to neurons

Besides the conversion of fibroblasts into blood cells and cardiomyocytes they have also been converted into neurons. Vierbuchen et al have identified Ascl1, Brn2 and Myt1l as the transcription factors that can convert both embryonic and postnatal fibroblasts into functional neurons.

The conversion was fast as at only 3 days after infection using lentiviruses they observed cells with a morphology similar to neurons. Within 12 days after infection they were able to detect induced neuron cells that expressed the pan-neuronal markers MAP2, NeuN and synapsin. The efficiency of the conversion ranged between 1,8% and 7,7%. They were able to detect glutamatergic neurons and GABA neurons. Either no expression of serotonin was detected.

Furthermore they studied the functionality of the neurons by focusing on the ability to form functional synapses. They reported that the induced neurons were able to form synapses and receive synaptic inputs from cortical neurons. The majority of induced neurons were excitatory. (Vierbuchen et al., 2010)



Aim of this review to answer the question: Is it possible to perform transdifferentiation of fibroblasts into fully functional somatic cells of a different lineage?

What methods are used to construct transdifferentiated cells? How do these differ with "conventional" embryonic or iPS techniques?

Technique of transdifferentiation is similar to that of generating iPS.

Ectopic expression using viral transfection.

Recipe of genes for different types of cells. Depending on target, specific recipe is used.

Are the resulting cells identical to the intended normal cells? What type of controls and tests are done to confirm?


Cell surface markers, gene expression profiles, expression of typical proteins

Cardiomyocytes: electrical conduction, Ca2+ influx

Neurons: synaps formation and neurotransmitter release

Macrophage, fagocytosis

How would transdifferentiated cells help in regenerative medicine? Are there any possible complications involved?

Viral transfection

Teratoma formation

Future clinical applications of stem cells concern a broad number of degenerative diseases (i.e. disease in which one cell type or part of an organ fails) which could potentially be treated using stem-cell-based therapy. This includes major metabolic diseases such as T1D (Type 1 diabetes), caused by the destruction of insulin-secreting β-cells [2], diverse brain and myelin disorders in which speci¬c neural cells are targeted such as MS (multiple sclerosis), PD (Parkinson's disease) or HD(Huntington's disease), heart disease, where some cardiac cells need to be replaced upon myocardial infarction, and genetic diseases like myopathy, where a speci¬c subtype of cells are not functional. The number of diseases that could be cured using stem cells is extremely broad.

Future research possibilities

Challenges to overcome

Alternatives for viral transfection.

Have some hurdles generated by embryonic and/or adult stem cells or iPS cells been successfully overcome by the technology of direct Transdifferentiation.

Unanswered questions

What is the underlying molecular mechanism? What is the role of epigenetics.

Despite years of research, relatively little is known about how lineage reprogramming is accomplished.

Final conclusion:

Certainly many potential, but it's still years away of it being used in the clinic.