BACKGROUND
cell therapy is now a viable alternative to liver transplantation for the treatment of chronic liver diseases.
however, this approach is limited by the shortage of reproducible sources of human hepatocytes  <cit> .
human pluripotent stem cells  are an ideal source for generating hepatocytes because they can be isolated, expanded as clonal populations to generate sufficient numbers, and differentiated in vitro.
human embryonic stem cells  remain the most reliable option, as they display an unlimited capacity for self-renewal.
we and others have generated hepatocyte-like cells from hescs in animal-free conditions by recapitulating liver developmental stages  <cit> .
however, although these differentiation protocols are relatively efficient, the presence of cells of an undesirable phenotype might pose health risks in the context of cell transplantation.
hence, for clinical applications, it is essential to transplant homogenous cell preparations that are highly enriched in the cells of interest, using a simple and reproducible procedure.
purified epithelial cell adhesion molecule epcam-positive cells from fetal and postnatal livers have been used to generate mature hepatocytes  <cit> , but this marker is also expressed in the visceral endoderm and in several progenitor cell populations and cancers, and is associated with undifferentiated hescs  <cit> .
a cell surface marker specific to hepatic progenitors that could be used for the simple and efficient fluorescence-activated cell sorting  of hepatic progenitors differentiated from hescs has not yet been identified.
alternative approaches based on the use of conventional lentiviral vectors  are complicated by the problem of genomic integration of transgenes and viral dna elements, potentially precluding their use for clinical applications.
however, integrase-defective lentivectors  can be produced by introducing a mutation into the integrase gene, which specifically prevents lentivector dna integration  <cit> .
transduction with idlvs results in the generation of circular vector episomes, and the transgene is expressed from these non-integrated proviral forms, which are progressively lost in proliferating cells, resulting in transient gene expression.
in a previous study, we designed a third-generation integrating lentivector  in which the gene encoding for green fluorescent protein  was under the control of the human liver-specific apoa-ii promoter.
we previously showed that this transgene is expressed in transduced primary simian hepatocytes both in vitro and in vivo after the transplantation of these transduced cells into animal models  <cit> .
by combining 1) cell sorting using a hepatic-specific promoter, 2) high-titer preparations of purified ilvs and idlvs, and 3) a specific integrase inhibitor, we created a robust and highly efficient method for purifying hesc-derived hepatic progenitors devoid of dna integration.
RESULTS
hepatic specificity of reporter lentivector expression
we first investigated the specificity of the apoa-ii promoter by transducing various cell lines with apoa-ii-gfp lentivector .
whereas the ubiquitous elongation factor 1α promoter was expressed in all cell lines tested, the apoa-ii promoter induced high levels of gfp expression only in the hepatic cell line huh <dig>  gfp expression was not detected in the human epithelial cell lines tested  nor in the cop cell line derived from human pancreatic islet cells, which like hepatic cells, are of endoderm origin .
because a meso-endoderm stage is common to both mesoderm and endoderm, we also verified the specificity of the apoa-ii promoter in endothelial cells , primary human fibroblasts , and primary mesenchymal stem cells  .
figure 1c shows a representative facs analysis of primary fibroblasts transduced with either the elongation factor 1α-gfp lentivirus or the apoa-ii-gfp lentivirus.
undifferentiated h <dig> cells transduced with apoa-ii-gfp vectors at a multiplicity of infection  of  <dig> displayed normal hesc morphology  and karyotype  and, as expected, did not express gfp .
purification of hepatic progenitors by facs
to assess the suitability of the apoa-ii reporter vector for a cell-sorting strategy, we directed the differentiation of transduced es cells into hepatic progenitors  <cit> , and the expression of apoa-ii-gfp was investigated during the various stages of cell differentiation.
as expected, hepatoblasts  expressed hepatic progenitor markers, such as α-fetoprotein , cytokeratin  <dig> , hepatic nuclear factor 4α and hnf <dig> .
from day  <dig> to day  <dig>  early markers of differentiation, such as sex determining region y-box  <dig> and hnf4α, were sequentially detected but less than 3% of the cells expressed gfp, reflecting the low level of apoa-ii promoter activity at this stage .
on day  <dig> , up to 39% of the cells expressed gfp .
sorting at day  <dig> yielded a population highly enriched in apoa-ii-gfp-expressing cells  .
after sorting, gfp-positive cells were plated on type i collagen-coated plates and maintained in culture.
after a 48-hour period, purified gfp-positive cells were homogeneously distributed, displayed the morphology of hepatic progenitors , and expressed both ck <dig> and afp .
generation of a purified population of hepatic progenitors devoid of virus integration
use of a conventional lentivector would result in major changes to the genetic material of the host genome, including potentially harmful mutations.
therefore, we aimed to develop a cell-purification strategy that would prevent permanent genome modification.
ef1α-gfp and apoa-ii-gfp were produced in an integration-defective form, with a gag/pol packaging plasmid encoding the d64v mutant integrase  <cit> .
we developed a protocol by transducing h <dig> cells with ef1α-gfp-idlv at various time points ,  <dig>  and 13) of the differentiation protocol, and monitoring the kinetics of gfp expression from days  <dig> to  <dig> after transduction.
the percentage of gfp-positive cells was highest on day  <dig> after transduction , with 60% of the cells expressing gfp at an moi of  <dig> when cells were transduced on day  <dig> of differentiation.
therefore, for subsequent experiments, we transduced hepatic cells on day  <dig> and sorted the fluorescent cell population  <dig> days later, that is, on day  <dig> of differentiation.
cells were transduced with purified ef1α-gfp-ilv and ef1α-gfp-idlv to confirm the advantages of idlv over ilv for sorting.
on day  <dig> after transduction, the proportions of gfp-idlv cells and gfp-ilv cells were similar, owing to episome transcription .
however, the proportion of gfp-idlv cells subsequently decreased to 1%  <dig> days after transduction, whereas the proportion of gfp-ilv-positive cells remained stable  as expected .
we also investigated the benefits of using raltegravir, an integrase inhibitor used in the clinical treatment of hiv infection, during the transduction protocol to prevent any residual vector integration  <cit> .
addition of raltegravir had no effect on the percentages of gfp cells on day  <dig> after transduction for either type of vector, owing to the presence of non-integrated forms, suggesting that this inhibitor has no effect on differentiation and/or transduction efficacy.
on day  <dig> after transduction, the proportion of idlv-transduced gfp-positive cells had decreased to less than 1%, in the presence or absence of raltegravir.
by contrast, raltegravir decreased the fraction of gfp-ilv-positive cells to less than 1% .
the final protocol used for the purification of hepatic progenitors from hescs is depicted in figure 4a.
as reported for apoa-ii-gfp-ilv, about 39% of differentiated cells were positive for apoa-ii-gfp-idlv at the time of sorting .
after a 2-day culture period, quantitative reverse transcription--pcr of gfp-sorted cells confirmed enrichment of ck <dig> expression compared with qrt-pcr performed on the day of sorting .
several co-immunostaining experiments showed that these cells co-expressed ck <dig> and afp, although at different levels, confirming their hepatic progenitor phenotype .
differentiation of purified hepatic progenitors devoid of viral dna integration
after sorting, cells were allowed to reach confluence in a serum-free medium previously defined for the culture of fetal hepatic progenitors, and then they were cultured in hepatocyte culture medium supplemented with hepatocyte growth factor  and oncostatin m. on day  <dig> after sorting , we analyzed the gfp expression of the cells.
only a very small number of fluorescent cells were visible by fluorescence microscopy, and facs analysis confirmed that no more than  <dig> % of the cells were fluorescent, whereas at day  <dig>  up to 35% cells were transduced .
cells transduced with either ilv or idlv in the presence or absence of raltegravir were analyzed or passaged on day  <dig> of differentiation , and the presence of lentivector dna forms were analyzed using the described probe .
a band common to both integrated and two bands  specific for non integrated forms  circle of the lentivector dna were detected in all transduced cells .
at day  <dig> after transduction , lentivector dna could be detected only in cells transduced with ilv in the absence of raltegravir .
integrated viral dna was absent from cells transduced either with idlv or with idlv or ilv in the presence of raltegravir.
in purified cells at day  <dig> of differentiation , no integrated or episomal dna derived from lentivectors was detected .
qpcr of genomic dna confirmed the absence of viral dna from all samples on day  <dig> of differentiation, with the exception of cells transduced with ilv in the absence of raltegravir .
the threshold of detection was analyzed by qpcr using a clonal cell line control  which, after transduction with a gfp lentivirus, contained one lentiviral integration per cell .
at a dilution of  <dig> in  <dig> , integration of viral dna was  <dig> times higher than the background in control non-transduced cells, and it was  <dig> to  <dig>  time higher at dilutions of  <dig> in  <dig> ,  <dig> in  <dig> , and  <dig> in  <dig> .
thus, these studies established the limit of detection of an integration event of less than  <dig> in  <dig> .
we then investigated whether hepatic progenitors were able to differentiate further into more mature hepatocytes.
on day  <dig> after sorting , the human progenitor-derived hepatocytes had acquired a morphology resembling that of hepatocytes , and had the functional characteristics of mature human hepatocytes.
these cells could incorporate and export indocyanin green  , secrete albumin , express clotting factor ix  mrna , and store glycogen .
finally, to further evaluate the functionality of differentiating hepatocytes, we sought to visualize expression of the mature hepatocyte-specific cytochrome p <dig> 3a <dig> .
the cells were transduced on day  <dig> of differentiation  with a lentivector expressing gfp under the control of the cyp3a <dig> promoter, and analyzed for fluorescence on day  <dig>   <cit> .
treatment of transduced cells with rifampicin, an inducer of cyp3a <dig>  produced a modest increase in gfp-positive cells, both in proportion and in mean fluorescence intensity  suggesting that the cyp3a <dig> promoter is indeed regulated in these hepatocytes .
discussion
safe and efficient methods for the purification of progenitor cells are a prerequisite to allow use of differentiated hpscs for regenerative medicine in order to minimize risks of tumorigenesis in patients.
to date, strategies for purifying a given cell population have used either a cell surface protein specific for the target cell population, such as stage-specific embryonic antigen - <dig> for isolation of human multipotent cardiovascular progenitor cells  <cit> , or lentivectors expressing a reporter gene under the control of a specific promoter  <cit> .
in this study, we show for the first time that it is feasible to purify a population of hesc-derived hepatic progenitors that are devoid of viral integration and that can differentiate further into more mature hepatocyte-like cells.
our strategy was to engineer human hepatic progenitors generated from a hesc line to transiently express gfp under the control of liver-specific apoa-ii.
it was crucial to choose appropriate vectors that would exhibit high transduction efficiency at low moi and would result in a vector-free enriched cell population.
for this study, we developed a method for producing purified ilvs and idlvs at high titers in order to minimize any deleterious effects upon transduction of target cells  <cit> .
the apoa-ii gene is expressed in liver and intestine, and we previously constructed a lentivector in which we inserted apoa-ii regulatory sequences to drive gfp expression, and confirmed its functionality both in vitro and in vivo <cit> .
in the current study, to assess the specificity of our construct, various cell types were transduced, including human epithelial lines and different sources of mesoderm, such as primary human mscs and fibroblasts.
when driven by the apoa-ii promoter, gfp was highly expressed only in hepatoma cells, confirming the suitability of this tool for purification of progenitors of the hepatic lineage and not for cells from mesodermal origin.
of note, gfp was not expressed in transduced human primary fibroblasts.
bi-potent mesendoderm, which can give rise to both definitive endoderm and mesoderm lineages, and transient populations expressing markers of both lineages, have been visualized in vivo <cit> .
the weak gfp expression seen in a low percentage of differentiating hes cells increased during the differentiation protocol, confirming the progressive differentiation of endoderm cells into hepatic cells.
this was confirmed by the upregulation of hnf4α, a key hepatic transcription factor in hepatic progenitors  <cit> .
thus, our results showed that the cells generated in our culture system display the physiological regulation of a hepatic-specific promoter and also display markers of hepatoblasts.
such markers were first identified in developing mouse liver  <cit> , and their expression in human progenitors has been confirmed by several groups, including us  <cit> .
our results also show that our purification approach does not prevent the sorted hepatoblasts from differentiating further into more mature hepatocytes, able to express fix , export icg, secrete albumin, and express and regulate the cyp3a <dig> promoter.
the hepatocytes we generated were not as fully mature as adult hepatocytes, but to our knowledge, this stage has not yet been achieved with pluripotent stem cell-derived hepatocytes  <cit> .
hiv integrase catalyses the enzymatic reactions that result in the covalent integration of the viral dna into the host dna  <cit> .
hiv- <dig> reporter viruses harboring mutations of integrase active-site residues are unable to catalysz viral dna integration, but still yield a reproducible level of reporter gene expression from the non-integrated proviral forms via dna episomal forms.
these viral episomes are progressively lost in dividing cells, leaving only the background level associated with non-specific random integration.
two types of circular episomes with intact coding regions are also produced.
homologous recombination within the ltrs generates a circular episome with a single ltr .
non-homologous end joining of the linear episome results in a circular episome with two adjacent ltrs.
our results show that,  <dig> days after progenitor transduction, lentivector integration was undetectable.
these results are in agreement with those of mátrai et al., who infused idlvs into liver and provided molecular evidence that the background integration was not mediated by residual catalytic activity of the mutant integrase  <cit> .
inhibition of integration by specific integrase inhibitors irreversibly blocks hiv- <dig> replication.
we reasoned that, as an alternative to idlv, use of an hiv integrase inhibitor in combination with ilv should also prevent any integration events.
raltegravir, which specifically targets the strand transfer reaction, and is currently being used in clinical trials  <cit> , abolished vector integration in our ilv-transduced progenitors, showing that use of ilv in combination with hiv integrase inhibitor can be an alternative to idlv.
thus, our methods can be used to purify hepatic progenitors prior to their differentiation into hepatocytes, and offers the advantage that resulting cells are free of lentivector dna integration.
given that xenogenic reagents such as mouse embryonic fibroblasts  or matrigel  are incompatible with the use of hpscs to treat debilitating human diseases  <cit> , our system, combined with the establishment of synthetic matrices, should facilitate the development of protocols for the generation of mature hepatocytes for future clinical applications.
CONCLUSIONS
our approach, based on purified idlv, facilitates the generation of a population of purified hesc-derived hepatic progenitors, which are devoid of integrated viral dna, and can be subsequently differentiated to mature hepatocyte-like cells.
this method represents a technological advance for many areas of research because it can be adapted easily for the purification of stem cell-derived populations such as pancreatic or neuronal cells for therapeutic purposes and also for in vitro applications such as drug screening.

 cell therapy is now a viable alternative to liver transplantation for the treatment of chronic liver diseases. however, this approach is limited by the shortage of reproducible sources of human hepatocytes. human pluripotent stem cells are an ideal source for generating hepatocytes because they can be isolated, expanded as clonal populations to generate sufficient numbers, and differentiated in vitro. human embryonic stem cells remain the most reliable option, as they display an unlimited capacity for self-renewal. we and others have generated hepatocyte-like cells from hescs in animal-free conditions by recapitulating liver developmental stages. however, although these differentiation protocols are relatively efficient, the presence of cells of an undesirable phenotype might pose health risks in the context of cell transplantation. hence, for clinical applications, it is essential to transplant homogenous cell preparations that are highly enriched in the cells of interest, using a simple and reproducible procedure. purified epithelial cell adhesion molecule epcam-positive cells from fetal and postnatal livers have been used to generate mature hepatocytes, but this marker is also expressed in the visceral endoderm and in several progenitor cell populations and cancers, and is associated with undifferentiated hescs. a cell surface marker specific to hepatic progenitors that could be used for the simple and efficient fluorescence-activated cell sorting of hepatic progenitors differentiated from hescs has not yet been identified. alternative approaches based on the use of conventional lentiviral vectors are complicated by the problem of genomic integration of transgenes and viral dna elements, potentially precluding their use for clinical applications. however, integrase-defective lentivectors can be produced by introducing a mutation into the integrase gene, which specifically prevents lentivector dna integration. transduction with idlvs results in the generation of circular vector episomes, and the transgene is expressed from these non-integrated proviral forms, which are progressively lost in proliferating cells, resulting in transient gene expression. in a previous study, we designed a third-generation integrating lentivector in which the gene encoding for green fluorescent protein was under the control of the human liver-specific apoa-ii promoter. we previously showed that this transgene is expressed in transduced primary simian hepatocytes both in vitro and in vivo after the transplantation of these transduced cells into animal models. by combining 1) cell sorting using a hepatic-specific promoter, 2) high-titer preparations of purified ilvs and idlvs, and 3) a specific integrase inhibitor, we created a robust and highly efficient method for purifying hesc-derived hepatic progenitors devoid of dna integration.  hepatic specificity of reporter lentivector expression we first investigated the specificity of the apoa-ii promoter by transducing various cell lines with apoa-ii-gfp lentivector. whereas the ubiquitous elongation factor 1α promoter was expressed in all cell lines tested, the apoa-ii promoter induced high levels of gfp expression only in the hepatic cell line huh gfp expression was not detected in the human epithelial cell lines tested nor in the cop cell line derived from human pancreatic islet cells, which like hepatic cells, are of endoderm origin. because a meso-endoderm stage is common to both mesoderm and endoderm, we also verified the specificity of the apoa-ii promoter in endothelial cells, primary human fibroblasts, and primary mesenchymal stem cells. figure 1c shows a representative facs analysis of primary fibroblasts transduced with either the elongation factor 1α-gfp lentivirus or the apoa-ii-gfp lentivirus. undifferentiated h cells transduced with apoa-ii-gfp vectors at a multiplicity of infection of displayed normal hesc morphology and karyotype and, as expected, did not express gfp. purification of hepatic progenitors by facs to assess the suitability of the apoa-ii reporter vector for a cell-sorting strategy, we directed the differentiation of transduced es cells into hepatic progenitors, and the expression of apoa-ii-gfp was investigated during the various stages of cell differentiation. as expected, hepatoblasts expressed hepatic progenitor markers, such as α-fetoprotein, cytokeratin, hepatic nuclear factor 4α and hnf. from day to day early markers of differentiation, such as sex determining region y-box and hnf4α, were sequentially detected but less than 3% of the cells expressed gfp, reflecting the low level of apoa-ii promoter activity at this stage. on day, up to 39% of the cells expressed gfp. sorting at day yielded a population highly enriched in apoa-ii-gfp-expressing cells. after sorting, gfp-positive cells were plated on type i collagen-coated plates and maintained in culture. after a 48-hour period, purified gfp-positive cells were homogeneously distributed, displayed the morphology of hepatic progenitors, and expressed both ck and afp. generation of a purified population of hepatic progenitors devoid of virus integration use of a conventional lentivector would result in major changes to the genetic material of the host genome, including potentially harmful mutations. therefore, we aimed to develop a cell-purification strategy that would prevent permanent genome modification. ef1α-gfp and apoa-ii-gfp were produced in an integration-defective form, with a gag/pol packaging plasmid encoding the d64v mutant integrase. we developed a protocol by transducing h cells with ef1α-gfp-idlv at various time points, and 13) of the differentiation protocol, and monitoring the kinetics of gfp expression from days to after transduction. the percentage of gfp-positive cells was highest on day after transduction, with 60% of the cells expressing gfp at an moi of when cells were transduced on day of differentiation. therefore, for subsequent experiments, we transduced hepatic cells on day and sorted the fluorescent cell population days later, that is, on day of differentiation. cells were transduced with purified ef1α-gfp-ilv and ef1α-gfp-idlv to confirm the advantages of idlv over ilv for sorting. on day after transduction, the proportions of gfp-idlv cells and gfp-ilv cells were similar, owing to episome transcription. however, the proportion of gfp-idlv cells subsequently decreased to 1% days after transduction, whereas the proportion of gfp-ilv-positive cells remained stable as expected. we also investigated the benefits of using raltegravir, an integrase inhibitor used in the clinical treatment of hiv infection, during the transduction protocol to prevent any residual vector integration. addition of raltegravir had no effect on the percentages of gfp cells on day after transduction for either type of vector, owing to the presence of non-integrated forms, suggesting that this inhibitor has no effect on differentiation and/or transduction efficacy. on day after transduction, the proportion of idlv-transduced gfp-positive cells had decreased to less than 1%, in the presence or absence of raltegravir. by contrast, raltegravir decreased the fraction of gfp-ilv-positive cells to less than 1%. the final protocol used for the purification of hepatic progenitors from hescs is depicted in figure 4a. as reported for apoa-ii-gfp-ilv, about 39% of differentiated cells were positive for apoa-ii-gfp-idlv at the time of sorting. after a 2-day culture period, quantitative reverse transcription--pcr of gfp-sorted cells confirmed enrichment of ck expression compared with qrt-pcr performed on the day of sorting. several co-immunostaining experiments showed that these cells co-expressed ck and afp, although at different levels, confirming their hepatic progenitor phenotype. differentiation of purified hepatic progenitors devoid of viral dna integration after sorting, cells were allowed to reach confluence in a serum-free medium previously defined for the culture of fetal hepatic progenitors, and then they were cultured in hepatocyte culture medium supplemented with hepatocyte growth factor and oncostatin m. on day after sorting, we analyzed the gfp expression of the cells. only a very small number of fluorescent cells were visible by fluorescence microscopy, and facs analysis confirmed that no more than % of the cells were fluorescent, whereas at day up to 35% cells were transduced. cells transduced with either ilv or idlv in the presence or absence of raltegravir were analyzed or passaged on day of differentiation, and the presence of lentivector dna forms were analyzed using the described probe. a band common to both integrated and two bands specific for non integrated forms circle of the lentivector dna were detected in all transduced cells. at day after transduction, lentivector dna could be detected only in cells transduced with ilv in the absence of raltegravir. integrated viral dna was absent from cells transduced either with idlv or with idlv or ilv in the presence of raltegravir. in purified cells at day of differentiation, no integrated or episomal dna derived from lentivectors was detected. qpcr of genomic dna confirmed the absence of viral dna from all samples on day of differentiation, with the exception of cells transduced with ilv in the absence of raltegravir. the threshold of detection was analyzed by qpcr using a clonal cell line control which, after transduction with a gfp lentivirus, contained one lentiviral integration per cell. at a dilution of in, integration of viral dna was times higher than the background in control non-transduced cells, and it was to time higher at dilutions of in, in, and in. thus, these studies established the limit of detection of an integration event of less than in. we then investigated whether hepatic progenitors were able to differentiate further into more mature hepatocytes. on day after sorting, the human progenitor-derived hepatocytes had acquired a morphology resembling that of hepatocytes, and had the functional characteristics of mature human hepatocytes. these cells could incorporate and export indocyanin green, secrete albumin, express clotting factor ix mrna, and store glycogen. finally, to further evaluate the functionality of differentiating hepatocytes, we sought to visualize expression of the mature hepatocyte-specific cytochrome p 3a. the cells were transduced on day of differentiation with a lentivector expressing gfp under the control of the cyp3a promoter, and analyzed for fluorescence on day. treatment of transduced cells with rifampicin, an inducer of cyp3a produced a modest increase in gfp-positive cells, both in proportion and in mean fluorescence intensity suggesting that the cyp3a promoter is indeed regulated in these hepatocytes. discussion safe and efficient methods for the purification of progenitor cells are a prerequisite to allow use of differentiated hpscs for regenerative medicine in order to minimize risks of tumorigenesis in patients. to date, strategies for purifying a given cell population have used either a cell surface protein specific for the target cell population, such as stage-specific embryonic antigen - for isolation of human multipotent cardiovascular progenitor cells, or lentivectors expressing a reporter gene under the control of a specific promoter. in this study, we show for the first time that it is feasible to purify a population of hesc-derived hepatic progenitors that are devoid of viral integration and that can differentiate further into more mature hepatocyte-like cells. our strategy was to engineer human hepatic progenitors generated from a hesc line to transiently express gfp under the control of liver-specific apoa-ii. it was crucial to choose appropriate vectors that would exhibit high transduction efficiency at low moi and would result in a vector-free enriched cell population. for this study, we developed a method for producing purified ilvs and idlvs at high titers in order to minimize any deleterious effects upon transduction of target cells. the apoa-ii gene is expressed in liver and intestine, and we previously constructed a lentivector in which we inserted apoa-ii regulatory sequences to drive gfp expression, and confirmed its functionality both in vitro and in vivo. in the current study, to assess the specificity of our construct, various cell types were transduced, including human epithelial lines and different sources of mesoderm, such as primary human mscs and fibroblasts. when driven by the apoa-ii promoter, gfp was highly expressed only in hepatoma cells, confirming the suitability of this tool for purification of progenitors of the hepatic lineage and not for cells from mesodermal origin. of note, gfp was not expressed in transduced human primary fibroblasts. bi-potent mesendoderm, which can give rise to both definitive endoderm and mesoderm lineages, and transient populations expressing markers of both lineages, have been visualized in vivo. the weak gfp expression seen in a low percentage of differentiating hes cells increased during the differentiation protocol, confirming the progressive differentiation of endoderm cells into hepatic cells. this was confirmed by the upregulation of hnf4α, a key hepatic transcription factor in hepatic progenitors. thus, our results showed that the cells generated in our culture system display the physiological regulation of a hepatic-specific promoter and also display markers of hepatoblasts. such markers were first identified in developing mouse liver, and their expression in human progenitors has been confirmed by several groups, including us. our results also show that our purification approach does not prevent the sorted hepatoblasts from differentiating further into more mature hepatocytes, able to express fix, export icg, secrete albumin, and express and regulate the cyp3a promoter. the hepatocytes we generated were not as fully mature as adult hepatocytes, but to our knowledge, this stage has not yet been achieved with pluripotent stem cell-derived hepatocytes. hiv integrase catalyses the enzymatic reactions that result in the covalent integration of the viral dna into the host dna. hiv- reporter viruses harboring mutations of integrase active-site residues are unable to catalysz viral dna integration, but still yield a reproducible level of reporter gene expression from the non-integrated proviral forms via dna episomal forms. these viral episomes are progressively lost in dividing cells, leaving only the background level associated with non-specific random integration. two types of circular episomes with intact coding regions are also produced. homologous recombination within the ltrs generates a circular episome with a single ltr. non-homologous end joining of the linear episome results in a circular episome with two adjacent ltrs. our results show that, days after progenitor transduction, lentivector integration was undetectable. these results are in agreement with those of mátrai et al., who infused idlvs into liver and provided molecular evidence that the background integration was not mediated by residual catalytic activity of the mutant integrase. inhibition of integration by specific integrase inhibitors irreversibly blocks hiv- replication. we reasoned that, as an alternative to idlv, use of an hiv integrase inhibitor in combination with ilv should also prevent any integration events. raltegravir, which specifically targets the strand transfer reaction, and is currently being used in clinical trials, abolished vector integration in our ilv-transduced progenitors, showing that use of ilv in combination with hiv integrase inhibitor can be an alternative to idlv. thus, our methods can be used to purify hepatic progenitors prior to their differentiation into hepatocytes, and offers the advantage that resulting cells are free of lentivector dna integration. given that xenogenic reagents such as mouse embryonic fibroblasts or matrigel are incompatible with the use of hpscs to treat debilitating human diseases, our system, combined with the establishment of synthetic matrices, should facilitate the development of protocols for the generation of mature hepatocytes for future clinical applications.  our approach, based on purified idlv, facilitates the generation of a population of purified hesc-derived hepatic progenitors, which are devoid of integrated viral dna, and can be subsequently differentiated to mature hepatocyte-like cells. this method represents a technological advance for many areas of research because it can be adapted easily for the purification of stem cell-derived populations such as pancreatic or neuronal cells for therapeutic purposes and also for in vitro applications such as drug screening. 
we used lentivectors encoding green fluorescent protein driven by the liver-specific apoliprotein a-ii promoter to purify human hepatic progenitors. we evaluated both integrating and integration-defective lentivectors in combination with an hiv integrase inhibitor. a human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. human pluripotent stem cells hold great promise for applications in regenerative medicine. we have developed an effective strategy to purify human hepatic cells from cultures of differentiating hpscs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. however, the safety of cell therapy using differentiated hpsc derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. subsequently, cells were transduced and sorted at day of differentiation to obtain a cell population enriched in hepatic progenitor cells.
 human pluripotent stem cells hold great promise for applications in regenerative medicine. however, the safety of cell therapy using differentiated hpsc derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. to date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods.  we used lentivectors encoding green fluorescent protein driven by the liver-specific apoliprotein a-ii promoter to purify human hepatic progenitors. we evaluated both integrating and integration-defective lentivectors in combination with an hiv integrase inhibitor. a human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. subsequently, cells were transduced and sorted at day of differentiation to obtain a cell population enriched in hepatic progenitor cells. after sorting, more than 99% of these apoa-ii-gfp-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin when further cultured for days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. moreover, they were devoid of vector dna integration.  we have developed an effective strategy to purify human hepatic cells from cultures of differentiating hpscs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. the present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells. tissue-specific progenitor cellshepatoblastspurificationnon-integrating lentiviral vectors 