BACKGROUND
differentiation during development is normally viewed as a one way process from undifferentiated to more differentiated cells.
however, some lower vertebrates such as teleost fish and some amphibians are able to compensate for the loss of body parts by regenerating a nearly perfect copy of the original part by dedifferentiating cells in vivo to facilitate regeneration.
after the loss of an appendage undifferentiated, pluri- or multipotent cells from different origins accumulate at the damaged surface to form a regeneration blastema.
the blastema is formed after wound closure through dedifferentiation of at least three terminally differentiated cell types, fibroblasts  <cit> , keratinocytes  <cit>  and myotubes  <cit> .
endogenous stem cells like muscle satellite cells also seem to provide cells for the blastema  <cit> .
despite the heterogeneous origin of the blastema cells, histologically they appear as a homogeneous population of cells and therefore have been traditionally viewed as a single cell type.
this view however has been recently challenged  <cit> .
after blastema formation, a period of extensive proliferation of blastema cells follows, before the cells re-differentiate to produce all the different cell types for the tissues of the missing appendage.
in contrast, it only recently became possible to dedifferentiate or reprogram somatic cells to pluripotent cells in vitro  <cit> .
exposure to just four transcription factors  is enough to reprogram fibroblasts and many other differentiated cell types into induced pluripotent stem  cells  <cit> .
this raises the question whether the in vivo dedifferentiation or reprogramming seen during regeneration has similarities to the in vitro reprogramming of fibroblasts to ips cells.
we noticed that two of the reprogramming factors were expressed during xenopus limb regeneration.
furthermore, a recent publication presented evidence that the reprogramming factors c-myc, sox <dig> and klf <dig> were expressed during regeneration in newts  <cit> .
therefore we thought to investigate the similarities and differences on a more systematic and broader scale.
in this paper we explore the possibility of similarities between reprogramming and regeneration from a molecular point of view.
we chose two of the current regeneration models, zebrafish and xenopus for this purpose.
each model offers different opportunities and techniques that add to the general picture of blastema cell formation.
in particular we concentrated on caudal fin regeneration in zebrafish and limb and tail regeneration in xenopus.
while these three structures are very different anatomically it has been shown that the underlying molecular mechanism of regeneration is very similar  <cit> , therefore conservation of the differentiation status of the blastema cells of these three appendages is as well expected.
we focused on the studies of gene expression by quantitative real time polymerase chain reaction  of regenerating and non-regenerating tissue compared to an embryonic, pluripotent reference cell type, analysis of the blastema cell cycle by fluorescence-activated cell sorting  and a functional approach by knocking down pou5f1/oct <dig> and sox <dig> with morpholinos in the zebrafish caudal fin demonstrating that some of the core factors needed for reprogramming are present and required during regeneration.
RESULTS
expression of pluripotency associated markers
to determine whether there are any similarities between in vivo regeneration and reprogramming of fibroblast cells to ips cells we made two assumptions.
first, we treated the blastema as a homogeneous population of cells, as would be predicted if they dedifferentiated to a pluripotent state and second, we expected overall levels of pluripotency associated factors to increase as abundance of blastema cells increases up to a comparable expression level of a pluripotent reference cell.
to assess the differentiation status of blastema cells a panel, consisting of zebrafish and xenopus homologues of the genes most commonly used to characterize mouse or human es and ips cells, was put together.
the panel includes the four most commonly used reprogramming factors oct <dig> , sox <dig> , klf <dig> , c-myc  as well as sall <dig> , lin <dig>  α <dig>  3/ <dig> fucosyltransferase lewis <dig>  which is the homologue of stage-specific embryonic antigen- <dig>  and others .
the panel, however, does not include nanog, a main player for pluripotency acquisition  <cit> , since no homologue has been described either in zebrafish or xenopus.
for oct <dig>  four homologues have been described in xenopus  and it has been shown for three of them that they can rescue the phenotype of an oct4-/- knockout mouse embryonic stem  cell line  <cit> .
forward and reverse primers used in quantification of relative expression by qpcr of zebrafish and xenopus genes.
gene names and accession numbers are specified as well as gene function.
we first tested the panel of genes in a cell type representing the closest to a pluripotent cell type in these species.
since neither xenopus nor zebrafish es cell cultures have been properly established  <cit>  we used cells from early developmental stages of both species.
for xenopus, animal cap cells were chosen as the pluripotent cell type, as these cells are able to differentiate into many different cell types of all three germ layers in culture by over-expressing specific genes or by culturing the caps in different conditions  <cit> .
all but two of the pluripotency associated markers tested in xenopus were expressed in the animal cap cells indicating that a similar gene core network might be operational during early amphibian development to confer pluripotency  to that found in the maintenance of pluripotency of mouse or human es/ips cells  <cit> .
the exceptions were fut <dig>  the homologue of ssea <dig>  and oct <dig> which did not show expression above the rt- control.
to cover the complete process of xenopus limb and tail regeneration we chose four discrete time points that represent different stages of regeneration to assess the differentiation status of blastema cells: non-regenerating tissue ), blastema formation , blastema expansion  and blastema redifferentiation .
if cells acquired pluripotency during regeneration one could expect to see an increase in pluripotency associated markers either during blastema formation  or blastema expansion .
since, xenopus loses the capacity to regenerate its limbs during metamorphosis   <dig> to 63)  <cit> , we included regenerative incompetent limbs at st <dig> as a further control in our study.
the expression profile of the same factors in xenopus blastema cells showed some differences to animal cap cells.
out of  <dig> factors tested only six or seven were expressed in a limb or tail blastema, respectively and each of these factors was also expressed in both non-regenerating limbs  and regenerative incompetent limbs  .
of these seven genes, four  were expressed at lower levels in the blastema than in pluripotent cells, while sox <dig> and c-myc, two of the factors facilitating reprogramming, and fut- <dig>  an early marker in the reprogramming process, were expressed at similar or higher levels.
not expressed above rt- control were the homologues of oct <dig> , lin <dig>  cripto <dig> and gdf <dig> .
furthermore, none of the factors tested was upregulated during limb regeneration.
however, some tissue specific differences between tail and limbs were found.
two of the factors, sall <dig> and fut <dig>  displayed modest upregulation during tail blastema formation, but no statistically significant upregulation during limb regeneration even though both factors showed a similar expression wave over time in tail and limb regeneration.
.
to our surprise we also found some upregulation of sox <dig>  c-myc and fut <dig> in regenerative incompetent pseudo blastemas of st <dig> limbs that peaked at  <dig> dpa and dropped back to expression levels of non-regenerating  limbs by  <dig> dpa.
since this peak of expression was not seen with all the genes tested, we assumed that it is biologically real and not caused by a technical error.
furthermore, sall <dig> is also significantly upregulated at  <dig> dpa and  <dig> dpa in the regenerative incompetent limbs .
we do not know what the significance of these expression peaks are, however they are certainly not relevant for pluripotency in these regenerative incompetent pseudo blastemas.
a similar early peak of sox <dig> expression has also been found in the non-regenerating ventral iris during lens regeneration in newts  <cit> .
to test the quality of our dataset, three other genes that have been described as blastema markers , were tested and showed the expected upregulation of expression during the first  <dig> dpa  giving us confidence in the quality of the dataset  <cit> .
other studies have reported sox <dig> and c-myc expression in newt lens and limb regeneration  <cit> .
while these authors report upregulation of the two factors in newt limbs, we did not see a significant change in expression levels during regeneration in xenopus.
moreover, the two factors were also expressed in regenerative incompetent limbs in xenopus, with c-myc being expressed at much higher levels in the older limb than in the younger, regenerative competent limb.
for zebrafish, embryos at the oblong blastula stage  were chosen to represent the pluripotent cell type since this stage is considered optimal to try to derive zebrafish es cells  <cit> .
these blastula derived cells have been shown to be able to contribute towards the germ line if injected into zebrafish embryos and therefore to be pluripotent  <cit> .
in accordance, we found that all eight pluripotency related genes tested were expressed in these blastula cells , with three of them  expressed at significantly higher levels than in early regenerating fin cells.
the other five genes  were expressed at low levels, both in the embryonic cells and in non-regenerating fin, with only sox <dig> showing significantly higher expression in fin than in embryonic cells .
we noticed, though, much higher sox <dig> expression in a slightly younger blastula stage  but not in oblong stage.
in agreement with results obtained in xenopus, none of the eight pluripotency associated markers tested showed any upregulation during wound healing , blastema formation  or blastema outgrowth .
intriguingly, pou5f <dig> was not completely shut down in adult fins and was also expressed in the blastema, while no expression was found in blastemas of either newts or xenopus .
some of the fin blastema markers were unexpectedly highly expressed also at oblong stage .
these results demonstrate that expression of pluripotency associated genes alone does not indicate pluripotency of that cell, since all of these genes are also expressed in differentiated cells of the limb, tail and fin at similar levels.
to make sure we did not miss any relevant gene in our panel, we investigated the relative expression of pluripotency associated genes in previously published microarray data sets from regenerating xenopus limbs and cap cells and zebrafish caudal fin .
we found good agreement with our qpcr results in that the relative expression of pluripotency associated markers was generally higher in animal cap cells than in xenopus blastema cells and in particular they were not upregulated upon amputation in limb blastema cells compared to non-regenerating limbs.
one notable difference though is, that oct <dig> was found to be more highly expressed in animal cap cells, but similarly was not expressed or very lowly expressed in blastema cells.
the difference might originate from a suboptimal primer set used in qpcr analysis.
the zebrafish caudal fin qpcr results too, were in agreement with published microarray data from regenerating adult fins.
generally the stem cell markers were expressed both in regenerating and non-regenerating fins and none was upregulated in blastema cells, while the relative expression of blastema markers was higher in regenerating adult fins  <cit> .
this confirms that our qpcr results represent the general trend.
spatial expression pattern of pluripotency associated stem cell markers
for function, not only absolute expression level but also spatial distribution of a factor matters.
therefore, we investigated the spatial expression of some of the reprogramming and pluripotency associated factors in xenopus looking at the expression patterns of the three factors that showed the highest absolute real time expression .
in situ hybridization revealed that c-myc was most highly expressed in the non-regenerating xenopus tail and the tail blastema where it spread over the whole of the regeneration bud, including notochord, neural tube and mesenchyme .
the expression in the developing  and regenerating limb bud  expression of c-myc in developing st <dig> limbs  and amputated limbs at  <dig> dpa,  <dig> dpa and  <dig> dpa .
c-myc expression is diffuse and very weak in the developing limb bud and the blastema.
b-b"') expression of sall <dig> in developing st <dig>  and regenerating limbs at  <dig> dpa,  <dig> dpa,  <dig> dpa .
sall <dig> is expressed diffusely in the distal mesenchyme of the developing limb bud and after amputation in the whole of the blastema.
c-c"') expression of sox <dig> in developing  and regenerating  limbs.
sox <dig> is expressed proximal to the blastema in the posterior mesenchyme.
d) c-myc mrna is expressed in the entire regeneration bud of a  <dig> dpa tail and is much stronger than in limbs.
e) sall <dig> mrna expression in  <dig> dpa tail is mainly seen in the distal tip of the regenerating tail.
f) sox <dig> mrna expression in  <dig> dpa tail is confined to the neural tube.
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thin black line indicates amputation plane
the expression pattern of sall <dig> in developing and regenerating limb buds has been published previously  <cit>  and is included here because it is one of the most highly expressed pluripotency associated genes in regeneration blastemas.
we found sall <dig> was localised to the distal mesenchyme of the developing st <dig> limb bud  and was also strongly expressed in the mesenchyme of the regenerating limb , which was in agreement with the published expression pattern.
in the regeneration bud of the tail sall <dig> was mainly localized to the distal tip .
in contrast, sox <dig> showed a very distinct localization to a posterior region in the developing limb bud just proximal to the amputation plane .
in the regenerating limb sox <dig> seemed still confined to the same region in the distal stump and showed little or no expression in the blastema itself .
during collection of blastemas for qpcr this distal stump region was included since dedifferentiation is thought to happen in this distal zone.
during tail regeneration sox <dig> expression was confined to the neural tube .
the diffuse expression pattern seen for c-myc and sall <dig> is compatible with a model where c-myc and sall <dig> induce or facilitate dedifferentiation of the stump tissue and keep the cells in a less differentiated state.
however, the localised expression pattern seen for sox <dig> points more towards a role in the development of a specific tissue or cell type in the neural tube and limb bud.
cell cycle analysis in regenerating and non-regenerating fin cells versus embryonic cells
embryonic stem cells differ markedly from other cells in their cell cycle profile and regulation.
it has been shown that es cells and other pluripotent cells have much shorter gap phases  or lack them altogether.
these particular cell cycle properties of es cells have been connected with pluripotency maintenance  <cit> .
to compare cell cycle profiles of pluripotent blastomere cells and fin blastema cells, we performed a facs analysis of their cell cycle.
cell survival after blastomere and blastemal cell recovery was high in all samples, ranging from 90% to 98% .
in all samples most of the cells were in g0/g <dig>  the percentage of cells in g2/mitosis  was quite similar in the non-regenerating fin ), early regenerating fin  and in the embryo  but higher in  <dig> dpa blastema cells  where regenerative outgrowth is at its highest .
while facs profile of cells showed similar percentage of cells in g2/m at  <dig> hpa and  <dig> hpa, specific differences were observed in ph <dig> expression in localized zones when  <dig> hpa cells and  <dig> hpa blastemal cells were compared  showing that cells in the blastema were more actively dividing than in the non-regenerating fin.
on the other hand, histograms from flow cytometric analysis of hoechst-stained cells did not show similarities between facs cell profile in embryos and in the regenerating fin  indicating that blastemal cells are controlled by a somatic cell cycle and do not revert to an embryonic one.
knockdown of either pou5f <dig> or sox <dig> impairs fin regeneration
having shown the presence of the reprogramming factors, we wanted to test whether they were necessary for regeneration.
to test for functionality we carried out a morpholino knockdown in the zebrafish caudal fin using two published morpholinos which had been tested in the embryo for specificity and function.
we chose to use morpholino  <dig>  against the pou5f <dig> gene  <cit> .
pou mo has been shown to phenocopy the mutant strain spiel-ohne-grenzen , which has a mutation in the pou5f <dig> gene.
the sox <dig> mo has been previously tested during retina pattern formation in zebrafish  <cit> .
since we did not know what phenotypes to expect for pou mo and sox <dig> mo injected fin blastemas, we used msxb morpholino, which has a published phenotype in fin regeneration as a positive control.
the homeobox gene msxb, a frequently used blastema marker, has been shown to be indispensable for blastema growth with a morpholino knockdown method  <cit> .
if we were able to replicate the published msxb mo phenotype in the fin then we could have confidence in the phenotype from the pou mo and sox <dig> mo.
as a control for unspecific phenotypes induced by injection we injected the lineage tracer dextran and a pou mo with five mismatches .
as expected neither dextran nor pou 5-mis mo had an effect on fin outgrowth ).
our positive control, msxb mo, injected into a  <dig> dpa blastema inhibited fin regeneration to a similar extent as previously published  <cit>  .
when pou mo and sox <dig> mo were injected into the dorsal  <dig> dpa fin blastema an impairment of regeneration was observed in the dorsal fin .
while msxb mo reduced overall regrowth in the dorsal fin to 77% of ventral fin regrowth, pou mo and sox <dig> mo reduced dorsal regrowth over four days to 78% and 87%, respectively.
however, if regrowth was calculated for the last  <dig> h only, when morpholino was actively present, the inhibition was much more pronounced, with dorsal regrowth down to 22%, 33% and 58% for msxb mo, pou mo and sox <dig> mo, respectively.
this demonstrates that both pou5f <dig> and sox <dig> are not only present in the fin blastema but also necessary for regeneration at  <dig> dpa .
injection of msxb mo only showed a significant reduction of fin outgrowth if injected into a  <dig> dpa blastema in accordance with the observed increase in msxb expression around that time as detected by qpcr or in situ hybridization  <cit> .
reasoning that the reprogramming factors pou5f <dig> and sox <dig> might be already needed at an earlier time point during fin regeneration we injected morpholino at different times during blastema formation and regrowth.
injection of morpholino into  <dig> dpa fins had no effect on regeneration, though for technical reasons we were only able to inject much lower volumes than into blastemas themselves.
injection of sox <dig> mo into a  <dig> dpa regenerating fin also did not lead to a phenotype .
however, pou mo injected into  <dig> dpa blastema inhibited dorsal fin outgrowth by 40%.
if morpholinos were injected into  <dig> dpa fin blastemas again pou mo but not sox <dig> mo reduced fin outgrowth by a similar extent as at  <dig> dpa .
so while msxb and sox <dig> are only needed for fin regrowth at three days post amputation, pou5f <dig> has an additional function during the formation and outgrowth of the early blastema.
to see if the reduction in regrowth was due to cell death we performed terminal deoxynucleotidyl transferase-mediated dutp nick end labeling  staining  <dig> h after injecting pou mo into  <dig> dpa fin blastemas to visualise dying cells.
a count of defined areas of four different fins revealed no difference in numbers of dying cells between the dorsal and the ventral fin .
in contrast, when we analysed cell proliferation by ph <dig> immunostaining in  <dig> dpa fin blastemas injected with pou mo  <dig> h earlier, we could see a clear and significant reduction  in cell divisions in the dorsal fin in three out of four fins .
the difference between dorsal and ventral cell divisions is not as big as expected because ph <dig> immunostaining only marks actively dividing cells at one particular time point.
furthermore dorsal outgrowth seems to recover over time since dorsal and ventral regrowth differences become less pronounced  <dig> h after mo injection.
discussion
restricted dedifferentiation of blastema cells
appendage regeneration is a complex biological process that takes place only in a few lower vertebrates like teleost fish and some amphibians.
a key step in the replacement of the lost appendage is the formation of the blastema, a transient structure of undifferentiated and proliferating cells which gives rise to all the different cell types in the newly formed appendage.
however, how exactly blastema formation is achieved is still unknown.
the idea that the process of dedifferentiation of fibroblast cells to ips cells and dedifferentiation during in vivo regeneration is regulated by a similar mechanism is attractive but unexplored.
for that reason, expression profiles of pluripotency associated factors in blastemal cells were compared with expression profiles in pluripotent embryonic reference cells.
a hallmark of vertebrate epimorphic regeneration is the re-activation of genes that are active during embryonic development  <cit> .
we assumed that if blastema cells became pluripotent they would reactivate or upregulate the genes which are part of the pluripotency network, as is seen for ips cells  <cit> .
however, we found that blastemal cells had little in common with our pluripotent reference cells.
blastema cells neither upregulated any of the key transcription factors needed for induction of ips cells  as would be expected if they became pluripotent nor did they significantly upregulate many of the other transcription factors needed for self-renewal in ips cells.
furthermore, hierarchical clustering of the published global gene expression data for xenopus animal cap cells and blastema cells  <cit>  demonstrated that they exhibit a different expression pattern, that is, blastema cells clustered on a different tree branch than pluripotent cells .
in other aspects as well, blastema cells were distinct from pluripotent cells.
the analyses of the cell cycle showed that blastema cells have all the characteristics of somatic cells and cycle more slowly than pluripotent stem cells.
furthermore, evaluation of the expression pattern of the three most highly expressed genes during xenopus regeneration in the qpcr analysis revealed that one is not expressed uniformly in the blastema as would be expected if it had a role in conferring or maintaining pluripotency in these cells.
this evidence suggests that blastema cells are not pluripotent but at best only multipotent.
a similar conclusion was reached for newt blastema cells since neither oct <dig> nor nanog, two of the central factors in the network conferring pluripotency to ips cells, were expressed  <cit> .
additionally, a recently published paper implies that blastema cells have less differentiation potential than thought  <cit> .
kragl et al.
reported that the regeneration blastema in axolotl is a pool of heterogeneous progenitor cells with restricted developmental potential, despite the rather uniform and undifferentiated appearance of blastema cells.
by labelling specific tissues with embryonic tissue grafts marked with green fluorescent protein they could show that each tissue produces its own progenitor cells.
these progenitors do not cross tissue boundaries during regeneration but only regenerate its own tissue type, so muscle regenerates muscle and cartilage regenerates cartilage, with the only exception of dermis which is able to make cartilage and dermis but not muscle.
in accordance, a recent proteomics analysis of regenerating xenopus limb buds revealed a significant number of upregulated proteins in blastema cells are also found in one or more adult stem cell types  <cit> .
whether this means that mature cell types dedifferentiate to become more like adult stem cells or whether resident adult stem cells are activated to produce blastema cells with hallmarks of the cells of origin is unclear at the moment.
possible mechanism and function
despite the evidence that blastema cells are not pluripotent there are some strong similarities between blastema cells and ips cells.
blastema cells and their precursors express low levels of some or all of the key factors needed for reprogramming to ips cells.
we hypothesise that these low levels are sufficient to trigger a similar mechanism to reprogramming which then gets stalled in an early, partially reprogrammed state leading to a cell which is only multipotent.
such intermediate states are frequently found during reprogramming among ips colonies.
some inroads have been made in deciphering the mechanism of reprogramming by analysing this/these intermediate state/s .
the process of reprogramming is slow and takes one to two weeks.
some of the earliest markers that are upregulated during this process are alkaline phosphatase  and ssea <dig>  the ssea <dig> expressing cells then activate other pluripotency associated genes like oct <dig>  sox <dig>  nanog and tert only late in the process.
the two early markers  are also either expressed or upregulated during blastema formation in xenopus .
therefore it is plausible that these two genes might function in a similar way during dedifferentiation and blastema formation as during reprogramming by facilitating the action of the later factors.
in zebrafish, none of the pluripotency associated markers tested are completely shut off in the adult fin.
similarly in xenopus seven of the  <dig> genes looked at are already expressed in the developing limb bud or tail.
avoiding a complete shutdown of gene expression should facilitate reactivation or reemployment of these factors later on, for example, during regeneration.
here we showed that these factors are indeed not just present but also required for regeneration.
a knockdown of pou5f <dig> with a gene specific morpholino impaired fin regrowth at various time points during regeneration.
and likewise a second gene, sox <dig> was also necessary for regeneration but to a lesser extent.
from these results we conclude that, despite pou5f <dig> expression in zebrafish regenerating fin not reaching levels of a pluripotent cell, this gene is crucial for fin regeneration from the very early stages of regeneration onwards.
at this stage, however, we can only speculate what that function is, either during regeneration or in the adult fin.
it has been shown that the function of pou <dig> proteins is conserved between mouse and xenopus and that they regulate similar genes in es cells and early xenopus embryos  <cit> .
the authors concluded that the ability of oct <dig> to maintain es cell pluripotency is derived from the ancestral function of this class of proteins to maintain multipotency during early vertebrate development.
it is reasonable to assume that the low level of pou5f <dig> expression during regeneration is not enough to confer real pluripotency reverting the blastema cells to a completely reprogrammed state, however, that a synergistic effect of pou5f <dig> together with other pluripotency genes, could enable these cells to dedifferentiate and provide them with the multipotency required to give rise to certain cell types, allowing the reconstitution of the lost structure.
however, while pou5f1/oct <dig> is required for fin regeneration, it does not seem to be essential for blastema formation in general, since neither xenopus nor newt reactivate pou5f1/oct <dig> homologue expression in the blastema  <cit> .
but then again reprogramming of neural stem cells has also been achieved without oct <dig> as one of the reprogramming factors indicating that reprogramming can be successful without exogenous oct <dig>  <cit> .
in contrast to what was observed for pou5f <dig>  sox <dig> seems to be mainly required for regenerative outgrowth similarly to what has been observed for msxb.
taking the expression pattern of sox <dig> in limb and tail blastema and its role in fin outgrowth into account we speculate that sox <dig> could have a role in specifying early neural precursor cells that contribute to the innervation of the regenerate.
a role for sox <dig> in neurogenesis has been previously described in other species  <cit>  and in mouse es cells  <cit> .
furthermore, in a previous in vitro study we observed that sox <dig> is expressed in blastula cell colonies that will subsequently give rise to neuronal precursors .
additionally, limb and fin blastema outgrowth requires innervation to proliferate and replace structures  <cit> .
research into the mechanism behind reprogramming also demonstrated that the presence of the reprogramming factors alone was not sufficient for success since secondary ips cells harbouring dox-inducible transgenes were only induced at a rate of 2%  <cit> .
there are several lines of evidence that a correct stoichiometric mixture of the reprogramming factors is necessary for successful reprogramming and that more is not necessarily better.
leaving some of the factors out of the reprogramming cocktail can increase ips efficiency in cells with expression of these factors  <cit> .
others have found that in an all-in-one vector approach, the sequence of the factors is important, again pointing towards the importance of delivering the correct relative level for each reprogramming factor.
this could explain why low levels of the pluripotency associated factors might be sufficient to start the dedifferentiation/reprogramming process during blastema formation if the stoichiometric requirement is fulfilled.
furthermore, it could also account for why older, non-regenerating xenopus limbs will not initiate blastema formation despite expressing the same factors but at different and ineffective stoichiometric combination.
CONCLUSIONS
knocking down the expression of pou5f1/oct <dig> and sox <dig> with morpholinos resulted in an impairment of regeneration, indicating that, despite the low expression levels, these factors do have a functional role during epimorphic regeneration.
in the light of this functional evidence we make an associative link between partially reprogrammed ips cells and the half way state of blastema cells and suggest that the processes leading to these two cell types might be regulated by some common mechanisms.

 differentiation during development is normally viewed as a one way process from undifferentiated to more differentiated cells. however, some lower vertebrates such as teleost fish and some amphibians are able to compensate for the loss of body parts by regenerating a nearly perfect copy of the original part by dedifferentiating cells in vivo to facilitate regeneration. after the loss of an appendage undifferentiated, pluri- or multipotent cells from different origins accumulate at the damaged surface to form a regeneration blastema. the blastema is formed after wound closure through dedifferentiation of at least three terminally differentiated cell types, fibroblasts, keratinocytes and myotubes. endogenous stem cells like muscle satellite cells also seem to provide cells for the blastema. despite the heterogeneous origin of the blastema cells, histologically they appear as a homogeneous population of cells and therefore have been traditionally viewed as a single cell type. this view however has been recently challenged. after blastema formation, a period of extensive proliferation of blastema cells follows, before the cells re-differentiate to produce all the different cell types for the tissues of the missing appendage. in contrast, it only recently became possible to dedifferentiate or reprogram somatic cells to pluripotent cells in vitro. exposure to just four transcription factors is enough to reprogram fibroblasts and many other differentiated cell types into induced pluripotent stem cells. this raises the question whether the in vivo dedifferentiation or reprogramming seen during regeneration has similarities to the in vitro reprogramming of fibroblasts to ips cells. we noticed that two of the reprogramming factors were expressed during xenopus limb regeneration. furthermore, a recent publication presented evidence that the reprogramming factors c-myc, sox and klf were expressed during regeneration in newts. therefore we thought to investigate the similarities and differences on a more systematic and broader scale. in this paper we explore the possibility of similarities between reprogramming and regeneration from a molecular point of view. we chose two of the current regeneration models, zebrafish and xenopus for this purpose. each model offers different opportunities and techniques that add to the general picture of blastema cell formation. in particular we concentrated on caudal fin regeneration in zebrafish and limb and tail regeneration in xenopus. while these three structures are very different anatomically it has been shown that the underlying molecular mechanism of regeneration is very similar, therefore conservation of the differentiation status of the blastema cells of these three appendages is as well expected. we focused on the studies of gene expression by quantitative real time polymerase chain reaction of regenerating and non-regenerating tissue compared to an embryonic, pluripotent reference cell type, analysis of the blastema cell cycle by fluorescence-activated cell sorting and a functional approach by knocking down pou5f1/oct and sox with morpholinos in the zebrafish caudal fin demonstrating that some of the core factors needed for reprogramming are present and required during regeneration.  expression of pluripotency associated markers to determine whether there are any similarities between in vivo regeneration and reprogramming of fibroblast cells to ips cells we made two assumptions. first, we treated the blastema as a homogeneous population of cells, as would be predicted if they dedifferentiated to a pluripotent state and second, we expected overall levels of pluripotency associated factors to increase as abundance of blastema cells increases up to a comparable expression level of a pluripotent reference cell. to assess the differentiation status of blastema cells a panel, consisting of zebrafish and xenopus homologues of the genes most commonly used to characterize mouse or human es and ips cells, was put together. the panel includes the four most commonly used reprogramming factors oct, sox, klf, c-myc as well as sall, lin α 3/ fucosyltransferase lewis which is the homologue of stage-specific embryonic antigen- and others. the panel, however, does not include nanog, a main player for pluripotency acquisition, since no homologue has been described either in zebrafish or xenopus. for oct four homologues have been described in xenopus and it has been shown for three of them that they can rescue the phenotype of an oct4-/- knockout mouse embryonic stem cell line. forward and reverse primers used in quantification of relative expression by qpcr of zebrafish and xenopus genes. gene names and accession numbers are specified as well as gene function. we first tested the panel of genes in a cell type representing the closest to a pluripotent cell type in these species. since neither xenopus nor zebrafish es cell cultures have been properly established we used cells from early developmental stages of both species. for xenopus, animal cap cells were chosen as the pluripotent cell type, as these cells are able to differentiate into many different cell types of all three germ layers in culture by over-expressing specific genes or by culturing the caps in different conditions. all but two of the pluripotency associated markers tested in xenopus were expressed in the animal cap cells indicating that a similar gene core network might be operational during early amphibian development to confer pluripotency to that found in the maintenance of pluripotency of mouse or human es/ips cells. the exceptions were fut the homologue of ssea and oct which did not show expression above the rt- control. to cover the complete process of xenopus limb and tail regeneration we chose four discrete time points that represent different stages of regeneration to assess the differentiation status of blastema cells: non-regenerating tissue ), blastema formation, blastema expansion and blastema redifferentiation. if cells acquired pluripotency during regeneration one could expect to see an increase in pluripotency associated markers either during blastema formation or blastema expansion. since, xenopus loses the capacity to regenerate its limbs during metamorphosis to 63), we included regenerative incompetent limbs at st as a further control in our study. the expression profile of the same factors in xenopus blastema cells showed some differences to animal cap cells. out of factors tested only six or seven were expressed in a limb or tail blastema, respectively and each of these factors was also expressed in both non-regenerating limbs and regenerative incompetent limbs. of these seven genes, four were expressed at lower levels in the blastema than in pluripotent cells, while sox and c-myc, two of the factors facilitating reprogramming, and fut- an early marker in the reprogramming process, were expressed at similar or higher levels. not expressed above rt- control were the homologues of oct, lin cripto and gdf. furthermore, none of the factors tested was upregulated during limb regeneration. however, some tissue specific differences between tail and limbs were found. two of the factors, sall and fut displayed modest upregulation during tail blastema formation, but no statistically significant upregulation during limb regeneration even though both factors showed a similar expression wave over time in tail and limb regeneration.. to our surprise we also found some upregulation of sox c-myc and fut in regenerative incompetent pseudo blastemas of st limbs that peaked at dpa and dropped back to expression levels of non-regenerating limbs by dpa. since this peak of expression was not seen with all the genes tested, we assumed that it is biologically real and not caused by a technical error. furthermore, sall is also significantly upregulated at dpa and dpa in the regenerative incompetent limbs. we do not know what the significance of these expression peaks are, however they are certainly not relevant for pluripotency in these regenerative incompetent pseudo blastemas. a similar early peak of sox expression has also been found in the non-regenerating ventral iris during lens regeneration in newts. to test the quality of our dataset, three other genes that have been described as blastema markers, were tested and showed the expected upregulation of expression during the first dpa giving us confidence in the quality of the dataset. other studies have reported sox and c-myc expression in newt lens and limb regeneration. while these authors report upregulation of the two factors in newt limbs, we did not see a significant change in expression levels during regeneration in xenopus. moreover, the two factors were also expressed in regenerative incompetent limbs in xenopus, with c-myc being expressed at much higher levels in the older limb than in the younger, regenerative competent limb. for zebrafish, embryos at the oblong blastula stage were chosen to represent the pluripotent cell type since this stage is considered optimal to try to derive zebrafish es cells. these blastula derived cells have been shown to be able to contribute towards the germ line if injected into zebrafish embryos and therefore to be pluripotent. in accordance, we found that all eight pluripotency related genes tested were expressed in these blastula cells, with three of them expressed at significantly higher levels than in early regenerating fin cells. the other five genes were expressed at low levels, both in the embryonic cells and in non-regenerating fin, with only sox showing significantly higher expression in fin than in embryonic cells. we noticed, though, much higher sox expression in a slightly younger blastula stage but not in oblong stage. in agreement with results obtained in xenopus, none of the eight pluripotency associated markers tested showed any upregulation during wound healing, blastema formation or blastema outgrowth. intriguingly, pou5f was not completely shut down in adult fins and was also expressed in the blastema, while no expression was found in blastemas of either newts or xenopus. some of the fin blastema markers were unexpectedly highly expressed also at oblong stage. these results demonstrate that expression of pluripotency associated genes alone does not indicate pluripotency of that cell, since all of these genes are also expressed in differentiated cells of the limb, tail and fin at similar levels. to make sure we did not miss any relevant gene in our panel, we investigated the relative expression of pluripotency associated genes in previously published microarray data sets from regenerating xenopus limbs and cap cells and zebrafish caudal fin. we found good agreement with our qpcr results in that the relative expression of pluripotency associated markers was generally higher in animal cap cells than in xenopus blastema cells and in particular they were not upregulated upon amputation in limb blastema cells compared to non-regenerating limbs. one notable difference though is, that oct was found to be more highly expressed in animal cap cells, but similarly was not expressed or very lowly expressed in blastema cells. the difference might originate from a suboptimal primer set used in qpcr analysis. the zebrafish caudal fin qpcr results too, were in agreement with published microarray data from regenerating adult fins. generally the stem cell markers were expressed both in regenerating and non-regenerating fins and none was upregulated in blastema cells, while the relative expression of blastema markers was higher in regenerating adult fins. this confirms that our qpcr results represent the general trend. spatial expression pattern of pluripotency associated stem cell markers for function, not only absolute expression level but also spatial distribution of a factor matters. therefore, we investigated the spatial expression of some of the reprogramming and pluripotency associated factors in xenopus looking at the expression patterns of the three factors that showed the highest absolute real time expression. in situ hybridization revealed that c-myc was most highly expressed in the non-regenerating xenopus tail and the tail blastema where it spread over the whole of the regeneration bud, including notochord, neural tube and mesenchyme. the expression in the developing and regenerating limb bud expression of c-myc in developing st limbs and amputated limbs at dpa, dpa and dpa. c-myc expression is diffuse and very weak in the developing limb bud and the blastema. b-b"') expression of sall in developing st and regenerating limbs at dpa, dpa, dpa. sall is expressed diffusely in the distal mesenchyme of the developing limb bud and after amputation in the whole of the blastema. c-c"') expression of sox in developing and regenerating limbs. sox is expressed proximal to the blastema in the posterior mesenchyme. d) c-myc mrna is expressed in the entire regeneration bud of a dpa tail and is much stronger than in limbs. e) sall mrna expression in dpa tail is mainly seen in the distal tip of the regenerating tail. f) sox mrna expression in dpa tail is confined to the neural tube.. thin black line indicates amputation plane the expression pattern of sall in developing and regenerating limb buds has been published previously and is included here because it is one of the most highly expressed pluripotency associated genes in regeneration blastemas. we found sall was localised to the distal mesenchyme of the developing st limb bud and was also strongly expressed in the mesenchyme of the regenerating limb, which was in agreement with the published expression pattern. in the regeneration bud of the tail sall was mainly localized to the distal tip. in contrast, sox showed a very distinct localization to a posterior region in the developing limb bud just proximal to the amputation plane. in the regenerating limb sox seemed still confined to the same region in the distal stump and showed little or no expression in the blastema itself. during collection of blastemas for qpcr this distal stump region was included since dedifferentiation is thought to happen in this distal zone. during tail regeneration sox expression was confined to the neural tube. the diffuse expression pattern seen for c-myc and sall is compatible with a model where c-myc and sall induce or facilitate dedifferentiation of the stump tissue and keep the cells in a less differentiated state. however, the localised expression pattern seen for sox points more towards a role in the development of a specific tissue or cell type in the neural tube and limb bud. cell cycle analysis in regenerating and non-regenerating fin cells versus embryonic cells embryonic stem cells differ markedly from other cells in their cell cycle profile and regulation. it has been shown that es cells and other pluripotent cells have much shorter gap phases or lack them altogether. these particular cell cycle properties of es cells have been connected with pluripotency maintenance. to compare cell cycle profiles of pluripotent blastomere cells and fin blastema cells, we performed a facs analysis of their cell cycle. cell survival after blastomere and blastemal cell recovery was high in all samples, ranging from 90% to 98%. in all samples most of the cells were in g0/g the percentage of cells in g2/mitosis was quite similar in the non-regenerating fin ), early regenerating fin and in the embryo but higher in dpa blastema cells where regenerative outgrowth is at its highest. while facs profile of cells showed similar percentage of cells in g2/m at hpa and hpa, specific differences were observed in ph expression in localized zones when hpa cells and hpa blastemal cells were compared showing that cells in the blastema were more actively dividing than in the non-regenerating fin. on the other hand, histograms from flow cytometric analysis of hoechst-stained cells did not show similarities between facs cell profile in embryos and in the regenerating fin indicating that blastemal cells are controlled by a somatic cell cycle and do not revert to an embryonic one. knockdown of either pou5f or sox impairs fin regeneration having shown the presence of the reprogramming factors, we wanted to test whether they were necessary for regeneration. to test for functionality we carried out a morpholino knockdown in the zebrafish caudal fin using two published morpholinos which had been tested in the embryo for specificity and function. we chose to use morpholino against the pou5f gene. pou mo has been shown to phenocopy the mutant strain spiel-ohne-grenzen, which has a mutation in the pou5f gene. the sox mo has been previously tested during retina pattern formation in zebrafish. since we did not know what phenotypes to expect for pou mo and sox mo injected fin blastemas, we used msxb morpholino, which has a published phenotype in fin regeneration as a positive control. the homeobox gene msxb, a frequently used blastema marker, has been shown to be indispensable for blastema growth with a morpholino knockdown method. if we were able to replicate the published msxb mo phenotype in the fin then we could have confidence in the phenotype from the pou mo and sox mo. as a control for unspecific phenotypes induced by injection we injected the lineage tracer dextran and a pou mo with five mismatches. as expected neither dextran nor pou 5-mis mo had an effect on fin outgrowth ). our positive control, msxb mo, injected into a dpa blastema inhibited fin regeneration to a similar extent as previously published. when pou mo and sox mo were injected into the dorsal dpa fin blastema an impairment of regeneration was observed in the dorsal fin. while msxb mo reduced overall regrowth in the dorsal fin to 77% of ventral fin regrowth, pou mo and sox mo reduced dorsal regrowth over four days to 78% and 87%, respectively. however, if regrowth was calculated for the last h only, when morpholino was actively present, the inhibition was much more pronounced, with dorsal regrowth down to 22%, 33% and 58% for msxb mo, pou mo and sox mo, respectively. this demonstrates that both pou5f and sox are not only present in the fin blastema but also necessary for regeneration at dpa. injection of msxb mo only showed a significant reduction of fin outgrowth if injected into a dpa blastema in accordance with the observed increase in msxb expression around that time as detected by qpcr or in situ hybridization. reasoning that the reprogramming factors pou5f and sox might be already needed at an earlier time point during fin regeneration we injected morpholino at different times during blastema formation and regrowth. injection of morpholino into dpa fins had no effect on regeneration, though for technical reasons we were only able to inject much lower volumes than into blastemas themselves. injection of sox mo into a dpa regenerating fin also did not lead to a phenotype. however, pou mo injected into dpa blastema inhibited dorsal fin outgrowth by 40%. if morpholinos were injected into dpa fin blastemas again pou mo but not sox mo reduced fin outgrowth by a similar extent as at dpa. so while msxb and sox are only needed for fin regrowth at three days post amputation, pou5f has an additional function during the formation and outgrowth of the early blastema. to see if the reduction in regrowth was due to cell death we performed terminal deoxynucleotidyl transferase-mediated dutp nick end labeling staining h after injecting pou mo into dpa fin blastemas to visualise dying cells. a count of defined areas of four different fins revealed no difference in numbers of dying cells between the dorsal and the ventral fin. in contrast, when we analysed cell proliferation by ph immunostaining in dpa fin blastemas injected with pou mo h earlier, we could see a clear and significant reduction in cell divisions in the dorsal fin in three out of four fins. the difference between dorsal and ventral cell divisions is not as big as expected because ph immunostaining only marks actively dividing cells at one particular time point. furthermore dorsal outgrowth seems to recover over time since dorsal and ventral regrowth differences become less pronounced h after mo injection. discussion restricted dedifferentiation of blastema cells appendage regeneration is a complex biological process that takes place only in a few lower vertebrates like teleost fish and some amphibians. a key step in the replacement of the lost appendage is the formation of the blastema, a transient structure of undifferentiated and proliferating cells which gives rise to all the different cell types in the newly formed appendage. however, how exactly blastema formation is achieved is still unknown. the idea that the process of dedifferentiation of fibroblast cells to ips cells and dedifferentiation during in vivo regeneration is regulated by a similar mechanism is attractive but unexplored. for that reason, expression profiles of pluripotency associated factors in blastemal cells were compared with expression profiles in pluripotent embryonic reference cells. a hallmark of vertebrate epimorphic regeneration is the re-activation of genes that are active during embryonic development. we assumed that if blastema cells became pluripotent they would reactivate or upregulate the genes which are part of the pluripotency network, as is seen for ips cells. however, we found that blastemal cells had little in common with our pluripotent reference cells. blastema cells neither upregulated any of the key transcription factors needed for induction of ips cells as would be expected if they became pluripotent nor did they significantly upregulate many of the other transcription factors needed for self-renewal in ips cells. furthermore, hierarchical clustering of the published global gene expression data for xenopus animal cap cells and blastema cells demonstrated that they exhibit a different expression pattern, that is, blastema cells clustered on a different tree branch than pluripotent cells. in other aspects as well, blastema cells were distinct from pluripotent cells. the analyses of the cell cycle showed that blastema cells have all the characteristics of somatic cells and cycle more slowly than pluripotent stem cells. furthermore, evaluation of the expression pattern of the three most highly expressed genes during xenopus regeneration in the qpcr analysis revealed that one is not expressed uniformly in the blastema as would be expected if it had a role in conferring or maintaining pluripotency in these cells. this evidence suggests that blastema cells are not pluripotent but at best only multipotent. a similar conclusion was reached for newt blastema cells since neither oct nor nanog, two of the central factors in the network conferring pluripotency to ips cells, were expressed. additionally, a recently published paper implies that blastema cells have less differentiation potential than thought. kragl et al. reported that the regeneration blastema in axolotl is a pool of heterogeneous progenitor cells with restricted developmental potential, despite the rather uniform and undifferentiated appearance of blastema cells. by labelling specific tissues with embryonic tissue grafts marked with green fluorescent protein they could show that each tissue produces its own progenitor cells. these progenitors do not cross tissue boundaries during regeneration but only regenerate its own tissue type, so muscle regenerates muscle and cartilage regenerates cartilage, with the only exception of dermis which is able to make cartilage and dermis but not muscle. in accordance, a recent proteomics analysis of regenerating xenopus limb buds revealed a significant number of upregulated proteins in blastema cells are also found in one or more adult stem cell types. whether this means that mature cell types dedifferentiate to become more like adult stem cells or whether resident adult stem cells are activated to produce blastema cells with hallmarks of the cells of origin is unclear at the moment. possible mechanism and function despite the evidence that blastema cells are not pluripotent there are some strong similarities between blastema cells and ips cells. blastema cells and their precursors express low levels of some or all of the key factors needed for reprogramming to ips cells. we hypothesise that these low levels are sufficient to trigger a similar mechanism to reprogramming which then gets stalled in an early, partially reprogrammed state leading to a cell which is only multipotent. such intermediate states are frequently found during reprogramming among ips colonies. some inroads have been made in deciphering the mechanism of reprogramming by analysing this/these intermediate state/s. the process of reprogramming is slow and takes one to two weeks. some of the earliest markers that are upregulated during this process are alkaline phosphatase and ssea the ssea expressing cells then activate other pluripotency associated genes like oct sox nanog and tert only late in the process. the two early markers are also either expressed or upregulated during blastema formation in xenopus. therefore it is plausible that these two genes might function in a similar way during dedifferentiation and blastema formation as during reprogramming by facilitating the action of the later factors. in zebrafish, none of the pluripotency associated markers tested are completely shut off in the adult fin. similarly in xenopus seven of the genes looked at are already expressed in the developing limb bud or tail. avoiding a complete shutdown of gene expression should facilitate reactivation or reemployment of these factors later on, for example, during regeneration. here we showed that these factors are indeed not just present but also required for regeneration. a knockdown of pou5f with a gene specific morpholino impaired fin regrowth at various time points during regeneration. and likewise a second gene, sox was also necessary for regeneration but to a lesser extent. from these results we conclude that, despite pou5f expression in zebrafish regenerating fin not reaching levels of a pluripotent cell, this gene is crucial for fin regeneration from the very early stages of regeneration onwards. at this stage, however, we can only speculate what that function is, either during regeneration or in the adult fin. it has been shown that the function of pou proteins is conserved between mouse and xenopus and that they regulate similar genes in es cells and early xenopus embryos. the authors concluded that the ability of oct to maintain es cell pluripotency is derived from the ancestral function of this class of proteins to maintain multipotency during early vertebrate development. it is reasonable to assume that the low level of pou5f expression during regeneration is not enough to confer real pluripotency reverting the blastema cells to a completely reprogrammed state, however, that a synergistic effect of pou5f together with other pluripotency genes, could enable these cells to dedifferentiate and provide them with the multipotency required to give rise to certain cell types, allowing the reconstitution of the lost structure. however, while pou5f1/oct is required for fin regeneration, it does not seem to be essential for blastema formation in general, since neither xenopus nor newt reactivate pou5f1/oct homologue expression in the blastema. but then again reprogramming of neural stem cells has also been achieved without oct as one of the reprogramming factors indicating that reprogramming can be successful without exogenous oct. in contrast to what was observed for pou5f sox seems to be mainly required for regenerative outgrowth similarly to what has been observed for msxb. taking the expression pattern of sox in limb and tail blastema and its role in fin outgrowth into account we speculate that sox could have a role in specifying early neural precursor cells that contribute to the innervation of the regenerate. a role for sox in neurogenesis has been previously described in other species and in mouse es cells. furthermore, in a previous in vitro study we observed that sox is expressed in blastula cell colonies that will subsequently give rise to neuronal precursors. additionally, limb and fin blastema outgrowth requires innervation to proliferate and replace structures. research into the mechanism behind reprogramming also demonstrated that the presence of the reprogramming factors alone was not sufficient for success since secondary ips cells harbouring dox-inducible transgenes were only induced at a rate of 2%. there are several lines of evidence that a correct stoichiometric mixture of the reprogramming factors is necessary for successful reprogramming and that more is not necessarily better. leaving some of the factors out of the reprogramming cocktail can increase ips efficiency in cells with expression of these factors. others have found that in an all-in-one vector approach, the sequence of the factors is important, again pointing towards the importance of delivering the correct relative level for each reprogramming factor. this could explain why low levels of the pluripotency associated factors might be sufficient to start the dedifferentiation/reprogramming process during blastema formation if the stoichiometric requirement is fulfilled. furthermore, it could also account for why older, non-regenerating xenopus limbs will not initiate blastema formation despite expressing the same factors but at different and ineffective stoichiometric combination.  knocking down the expression of pou5f1/oct and sox with morpholinos resulted in an impairment of regeneration, indicating that, despite the low expression levels, these factors do have a functional role during epimorphic regeneration. in the light of this functional evidence we make an associative link between partially reprogrammed ips cells and the half way state of blastema cells and suggest that the processes leading to these two cell types might be regulated by some common mechanisms. 
dedifferentiation also occurs in the induction of pluripotent stem cells when a set of transcription factors is over expressed in mature cell types. we hypothesised that there are parallels between dedifferentiation or reprogramming of somatic cells to induced pluripotent stem cells and the natural process of dedifferentiation during epimorphic regeneration. we therefore propose a link between partially reprogrammed induced pluripotent stem cells and the half way state of blastema cells and suggest that a common mechanism might be regulating these two processes. dedifferentiation occurs naturally in mature cell types during epimorphic regeneration in fish and some amphibians. however, during blastema formation some of the key reprogramming factors are both expressed and are also required for regeneration to take place. by comparing cells from the regeneration blastema with embryonic pluripotent reference cells we found that induced pluripotent stem and blastema cells do not share pluripotency. we analysed expression levels of the most commonly used pluripotency associated factors in regenerating and non-regenerating tissue and compared them with levels in a pluripotent reference cell. however these factors were not upregulated during regeneration as would be expected if blastema cells acquired pluripotency.
 dedifferentiation occurs naturally in mature cell types during epimorphic regeneration in fish and some amphibians. dedifferentiation also occurs in the induction of pluripotent stem cells when a set of transcription factors is over expressed in mature cell types.  we hypothesised that there are parallels between dedifferentiation or reprogramming of somatic cells to induced pluripotent stem cells and the natural process of dedifferentiation during epimorphic regeneration. we analysed expression levels of the most commonly used pluripotency associated factors in regenerating and non-regenerating tissue and compared them with levels in a pluripotent reference cell. we found that some of the pluripotency associated factors were expressed before and during regeneration and that at least two of these factors were also required for normal fin regeneration in the zebrafish. however these factors were not upregulated during regeneration as would be expected if blastema cells acquired pluripotency.  by comparing cells from the regeneration blastema with embryonic pluripotent reference cells we found that induced pluripotent stem and blastema cells do not share pluripotency. however, during blastema formation some of the key reprogramming factors are both expressed and are also required for regeneration to take place. we therefore propose a link between partially reprogrammed induced pluripotent stem cells and the half way state of blastema cells and suggest that a common mechanism might be regulating these two processes. 