the various blood cell lineages in mammals arise from a multipotent haematopoietic stem cell via particular differentiation pathways .
one of these pathways , erythropoiesis , leads to the production of red blood cells ( rbcs ) , which transport oxygen and carbon dioxide around the body by means of the intracellular metalloprotein haemoglobin ( hb ) .
hb is a tetramer , consisting of two -globin and two -globin subunits . in mammals ,
these globin chains are encoded by two gene loci : the -globin locus and the -globin locus . in humans ,
the -globin locus consists of the embryonic - and adult -globin genes , and the -globin locus comprises the embryonic - , foetal - and - , and adult - and -globin genes     [ 1 , 2 ] .
the globin genes expressed from these loci differ from embryonic to adult erythropoiesis in order to meet varying oxygen demands and facilitate placental transfer of oxygen from mother to embryo .  
there are a number of severe diseases caused by the disruption of adult globin genes , including thalassaemias and certain types of anaemia . according to the world health organisation ,
approximately 5% of the world 's population carry genes involved in hb disorders , and as such , they present an enormous health burden .
thalassaemia is caused by a reduction or abolition of the expression of one or more globin genes , resulting in an imbalance of - and -globin chains in red blood cells and consequent anaemia     [ 1 , 4 ] .
sickle cell anaemia is another prevalent haemoglobinopathy and is caused by a mutation in the adult -globin gene which generates a single glutamic acid to valine amino acid substitution .
this mutation leads to the polymerisation of globins in venous circulation     [ 5 , 6 ] , which can trigger a rigid and sickled cell phenotype     [ 7 , 8 ] and results in a number of acute conditions such as vaso - occlusion , splenic sequestration , and haemolytic anaemia .  
there are currently a number of treatments available for patients suffering from thalassaemia and sickle cell anaemia .
the most common is packed red blood cell transfusion , but this is associated with a number of problems , such as sufficiency of supply , bacterial and viral infection , biochemical and biomechanical changes during storage ( red blood cell storage lesions ) , and the risk of immune rejection from the patient     [ 10 , 11 ] .
furthermore , blood transfusions are ongoing throughout a patient 's life and often lead to a potentially fatal buildup of iron and associated reduction in organ activity .  
residual production of foetal -globin persists naturally throughout life , and levels vary between individuals     [ 12 , 13 ] .
this persistent expression allows two -globin chains to combine with two adult -globin chains to form what is known as foetal hb ( hbf ) . as only the adult -globin gene
is mutated in sickle cell anaemia , affected infants are protected from severe symptoms until they reach several months of age , due to the large amount of hbf still in circulation at birth .
furthermore , patients who have inherited alleles associated with increased levels of hbf , known as hereditary persistence of foetal hb ( hpfh ) , are protected into adulthood . similarly , a more asymptomatic disease phenotype has also been shown in patients with -thalassaemia who exhibit higher levels of hbf     .
together , these observations indicate that increased foetal -globin is able to compensate in part for the loss of adult -globin function and thus ameliorates the symptoms of certain adult haemoglobinopathies .
accordingly ,   a number of drug treatments for -thalassaemia and sickle cell disease , for instance , 5-azacitidine , hydroxyurea , and butyrate , all act by nonspecifically reactivating foetal -globin gene expression by various mechanisms .
there is evidence that long - term administration of these drugs has chronic side effects , consistent with their lack of specificity     [ 8 , 17 ] .   as the existing methods of treatment for these haemoglobinopathies remain inadequate ,
alternative forms of therapy are currently being sought , and stem cell therapies should be considered .
this paper will discuss progress in utilising novel cellular reprogramming techniques to treat rbc diseases .
stem cells , both embryonic and adult , have the ability to differentiate into various cell types , making them a potentially attractive treatment option . embryonic stem cells ( escs ) and adult stem cells ( ascs ) each have their own strengths and disadvantages in these strategies .
escs are more easily grown in culture and are pluripotent , meaning that they are able to differentiate into any cell of the body .
the practicality of widespread esc use for therapeutic purposes , however , has been questioned due to issues of supply and ethical and legal considerations . moreover
ascs , on the other hand , overcome some of these problems as they can be harvested from each individual patient .
they are not abundant and are difficult to obtain , often being harboured in internal organs such as the gut and bone marrow .
they have proven difficult to culture in vitro , and furthermore , they are widely believed to be multipotent rather than pluripotent and are thus only able to differentiate into certain cell types .
cellular reprogramming potentially overcomes these issues and may offer new treatment methods for a range of diseases , including those of rbcs . takahashi and yamanaka      were the first to show that it is possible to take differentiated somatic cells and transform them into cells with pluripotent potential .
they began by identifying a pool of 24 transcription factors which are important in maintaining stem cell traits and used retroviral transduction to express these factors in murine embryonic and adult fibroblasts .
they found that these cells then displayed characteristics and properties comparable to those of pluripotent escs .
they were able to further refine the required transcription factors by setting up numerous combinations to determine which were essential to this process and identified four factors , oct4 , sox2 , klf4 , and c - myc , needed to transform a somatic cell to an induced pluripotent stem cell ( ipsc ) .
takahashi et al .      then applied these four factors to human cells and transformed neonatal and adult human fibroblasts into human ipscs ( hipscs ) .
this was a valuable subsequent step as it showed that this process can be reproduced with human cells and could thus potentially be utilised for stem cell - based therapies of human disease .
several other transcription factor combinations have since been shown to be sufficient to reprogram somatic cells     [ 2224 ] and one particular combination , involving oct4 , sox2 , nanog , and lin28 , is particularly efficient and is now widely utilised .  
for example , a number of the transcription factors used to generate these cells , including c - myc , klf4 , and lin28 , are oncogenes , and their misexpression can lead to cancer     [ 2628 ] . in order for ipscs to be differentiated into a cell type of choice ,
the retrovirally transmitted genes need to be switched off or removed to reduce the possibility of their inducing tumours [ 22 , 29 ] .
another potential issue is that the transplantation of any cells that have not been fully differentiated from the ipsc state could lead to the formation of cancerous teratomas .
any cells remaining which are still in a pluripotent state could multiply and , without the appropriate growth controls , would have the potential to result in tumours in transplant patients . to potentially avoid the issue of tumour formation and uncontrolled proliferation of ipscs , an alternative methodology known as transdifferentiation is also being considered as a potential treatment strategy .  
transdifferentiation is achieved by introducing various exogenous factors into a differentiated cell , such as a fibroblast , to directly convert it into another type of differentiated cell , thereby bypassing the pluripotent state ( figure 1(b ) ) . as early as 1990 , choi et al .   were able to convert various cells types , including dermal fibroblasts and chondroblasts , into mononucleated , striated myoblasts that were indistinguishable from normal myoblasts in vivo
this was achieved through the expression of the myogenic regulatory factor myod , a transcription factor known to be involved in the determination of muscle cells . shortly after this
, another group showed that it was possible to turn myeloid 416b cells into mast cells through the forced expression of gata-2 and gata-3 , two transcription factors which play important roles in haematopoiesis .
investigated the effects of ectopically expressing the gene eyeless ( ey ) , an orthologue of the mammalian pax6 gene , in drosophila and found that eye structures formed in places such as the wings and legs .  
since takahashi and yamanaka 's pioneering studies in cellular reprogramming , the field of transdifferentiation has advanced considerably .
zhou et al .   investigated the effects of expressing ngn3 , pdx1 , and mafa , transcription factors involved in -cell differentiation , on exocrine cells of the adult pancreas .
they found that the coexpression of these factors was able to convert the exocrine cells into -cells .
the induced -cells were identical to endogenous -cells in morphology and showed similar expression of genes associated with -cell function .
these cells can also rescue the phenotype of hyperglycaemia , as they are able to secrete insulin and remodel surrounding vasculature . in other work ,
utilised the neural - specific transcription factors , ascl1 , brn2 , and myt1l , to rapidly convert both murine embryonic fibroblasts and adult fibroblasts directly into functional neurons .
these induced neuronal cells express neuron - specific proteins , generate action potentials , and form functional synapses .
a further advance has shown that it is also possible to transdifferentiate fibroblasts into functional neural progenitor cells by transient induction of oct4 , sox2 , klf4 , and c - myc in cells cultured in a defined neural reprogramming medium . this process bypassed the generation of ipscs and gave rise to multipotent progenitors with the capacity to expand and differentiate into a number of neural lineages .
all of these experiments indicate that it is possible to direct a differentiated cell to another cell fate through the application of extragenic factors .
advances in cellular reprogramming have raised the attractive possibility that this technology could be utilised to generate a limitless source of immune - matched , pathogen - free erythrocytes for transfusion .
they found that hipscs are capable of generating haematopoietic cells with phenotypic and morphological characteristics similar to those derived from hescs ; however , these hipsc - derived cells exhibited a dramatically reduced capacity ( by greater than 1000-fold ) to generate erythroid cells .   a subsequent study by lapillone et al .
, however , showed that it was indeed possible to produce significant numbers of mature erythroid cells from hipscs in vitro .
this group employed the methods outlined by thomson 's group   using oct4 , sox2 , nanog , and lin28 to convert fibroblasts to   hipscs .
they then cultured these cells in medium containing the cytokines scf , tpo , flt3 ligand , rhu bmp4 , rhu vegf - a165 , il-3 , il-6 , and erythropoietin ( epo ) .
they analysed the expression profiles of these cells over 20 days of culture and found that pluripotent stem cell markers decreased whilst the erythroid markers cd36 , cd235a , and cd71 increased .
cells at day 20 were found to have a high erythroid potential and were plated in sequential cocktails of cytokines comprising scf , il-3 , and/or epo .
erythroid maturation was achieved after another 25 days and was confirmed by morphological examination and by flow cytometric analysis of erythroid markers ( cd235a , cd71 , and cd36 ) .
furthermore , these erythroid cells were able to enucleate , albeit with reduced capacity compared to hesc - derived erythroid cells , and were found to express functional hb ( predominantly hbf ) .
these hipsc - derived erythroid cells were compared to those differentiated from hescs , and no significant differences were detected in terms of erythroid commitment , expression of erythroid markers , and type and functionality of hb .  
revealed that while functional rbcs could be generated from hipscs , when compared to hescs , hipscs were shown to have reduced ( approximately 8-fold ) amplification potential in producing mature erythroid cells .
a recent study has set out to determine whether this reduced efficiency is an intrinsic property of hipscs , or whether it can be increased by altered culture conditions .
this study showed not only that it is possible to generate rbcs from hipscs with similar efficiency to hescs , but also that these cells can be differentiated from hipscs generated using episomal vectors , obviating the need for potentially deleterious retroviral transfection .
this group cultured hipscs derived from human fibroblasts with the op9 bone marrow stroma line to induce hematopoietic differentiation and followed this with selective expansion of erythroid cells in serum - free media with cytokines supporting rbc differentiation .
the erythroid cultures established in this study consisted of a pure population of cd235a+ cd45 leukocyte - free rbcs which had robust expansion capabilities , as well as a long lifespan of up to 90 days .
these hipsc - derived cells can enucleate and were shown to express foetal - and embryonic -globin demonstrating successful reprogramming of the -globin locus .
the results from this study show that it is possible to produce significant numbers of erythroid cells from fibroblast - derived hipscs , and that thus cellular reprogramming could contribute to the treatment of haemoglobinopathies . furthermore , since these rbcs are generated from transgene - free hipscs , they circumvent problems associated with genomic integration and undesired reactivation of reprogramming factors .   another recent study has shown that it is possible to use transdifferentiation to produce haematopoietic cell lineages , including mature erythrocytes . in this work , szabo et al .
transduced human dermal fibroblasts with oct4 alone and showed that these cells then express the panhaematopoietic marker cd45 but lack the pluripotency marker tra-1 - 60 .
they then cultured these cells in a haematopoietic cytokine cocktail containing scf , g - csf , flt3lg , il-3 , il-6 , and bmp-4   supplemented with epo and observed an emergence of cells expressing adult -globin protein and the red blood cell marker glycophorin - a .
in addition , the authors noted that these erythroid cells express adult rather than embryonic globins , unlike cells derived from human pluripotent stem cells     [ 39 , 40 ] , perhaps suggesting that they have indeed been transdifferentiated from adult fibroblasts , bypassing the hipsc stage .
as the techniques used in this study avoid the pluripotent state as well as have a high yield , expansion capacity , and clinical feasibility , this strategy could provide a reasonable basis for autologous cell replacement therapies .  
these studies demonstrate that it is possible to differentiate hipscs into mature erythrocytes in vitro for transfusions . in order to utilise these methods to treat rbc diseases successfully
, fibroblasts could potentially be taken from either a healthy immunomatched individual , or a universal donor , and be converted to erythrocytes via the hipsc state .
a study has already developed hipscs from an individual with a bombay phenotype of the abo blood group system , where the abh antigen is not expressed on rbcs , and blood can be donated to anyone     .
this group has further demonstrated that it is possible to differentiate haematopoietic lineages from these cells although they have not explicitly shown mature erythrocyte differentiation .
this possible treatment method circumvents many of the problems associated with current transfusions , such as questionable quality , lack of supply , and immune - rejection .
however , issues still remain , such as cost and the risk of iron overloading .
in order to cure , rather than treat , rbc diseases , healthy progenitor cells must be transplanted into patients and subsequently repopulate the haematopoietic system .
a promising study has already shown that it is possible to use reprogrammed cells to treat sickle cell anaemia in mice .
hanna et al .      took cells from the tail tips of a humanized sickle cell anaemic mouse model , in which the mouse -globin genes have been replaced with human -globin , and the mouse -globin genes have been replaced with human  and  ( sickle ) globin genes , and through adaptation of the protocol developed by takahashi and yamanaka , who reprogrammed these cells into ipscs .
once this was accomplished , they were able to correct the mutant -globin gene by replacing it with a copy of the wildtype human -globin gene through homologous recombination . following this ,
the ipscs were differentiated into haematopoietic progenitors through the ectopic expression of hoxb4 , and by growing in haematopoietic cytokines on an op9 bone marrow stroma cell line .
this group then carried out extensive testing to determine whether the treated mice displayed any further signs of the disease .
firstly , functional correction was evaluated by electrophoresis for the human -globin proteins  and  , and they found a significant increase in - and a decrease in -globin .  
blood counts were also performed 12 weeks after transplant , which showed that treated mice had increased rbcs , hb , and packed cell volume as well as reduced reticulocytes , a common indicator of sickle cell disease and severity .
lastly , they examined symptomatic indicators of sickle cell anaemia such as urine concentration , body weight , and breathing rate and found that all three of these parameters were ameliorated in the treated mice .
this study thus provides an important proof of principle that cellular reprogramming can be employed to correct erythroid disorders , albeit in this case , in conjunction with gene therapy .
normal erythropoiesis is dependent upon the correct expression of globin genes . where globin genes are incorrectly expressed , or are mutated , anaemia or thalassemia results .
current therapy for these disorders involves packed red blood cell transfusions , which are limited by supply , risk of infection , expense , and patient rejection .
drug - based therapies involve the nonspecific reactivation of foetal globins and have long - term side effects . in seeking alternative strategies
, recent advances have shown that cellular reprogramming can now generate large quantities of red blood cells in culture , potentially for use in transfusions .
furthermore , these strategies have been successfully combined with gene therapy to treat a sickle cell anaemia mouse model , suggesting that cellular reprogramming will provide a realistic future alternative to conventional treatment of haemoglobinopathies .
 haemoglobinopathies such as thalassaemia and sickle cell disease present a major health burden . 
 currently , the main forms of treatment for these diseases are packed red blood cell transfusions and the administration of drugs which act to nonspecifically reactivate the production of foetal haemoglobin . 
 these treatments are ongoing throughout the life of the patient and are associated with a number of risks , such as limitations in available blood for transfusion , infections , iron overload , immune rejection , and side effects associated with the drug treatments . 
 the field of cellular reprogramming has advanced significantly in the last few years and has recently culminated in the successful production of erythrocytes in culture . 
 this paper will discuss cellular reprogramming and its potential relevance to the treatment of haemoglobinopathies . 
1. Introduction: Globin Gene Regulation and Haemoglobinopathies
2. Cellular Reprogramming
3. Cellular Reprogramming as a Potential Treatment for Anaemia
4. Cellular Reprogramming as a Potential Cure for Anaemia
5. Conclusions