I ) Activation of pAKT ( Magenta ) was examined in Icos1ø and Icos1 treated - animals within or around vascular ( CD31 + , green ) and pericytes ( NG2 + cells , yellow ) of the BBB . High - resolution confocal images ( insets ) reveal the prevalence of activated pAKT ( magenta ) co - stain ( cyan arrows ) in pericytes ( yellow ) of the BBB along vasculature ( green , or dotted outline ) versus reactive neuroglia ( NG2 + pAKT - ) in the parenchyma ( red arrows ) in Icos1ø vs . Icos1 treated animals . Scale bar is 10 µm .

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I) Activation of pAKT (Magenta) was examined in Icos1ø and Icos1 treated-animals within or around vascular (CD31+, green) and pericytes (NG2+ cells, yellow) of the BBB. High-resolution confocal images (insets) reveal the prevalence of activated pAKT (magenta) co-stain (cyan arrows) in pericytes (yellow) of the BBB along vasculature (green, or dotted outline) versus reactive neuroglia (NG2+pAKT-) in the parenchyma (red arrows) in Icos1ø vs. Icos1 treated animals. Scale bar is 10 µm.


( A , B ) Flies form well - organized compound eyes when they carry GMR - GAL4 without a target gene ( A ) but develop rough eyes when GMR - GAL4 drives expression of UAS - Foxn1 ( B ) . Panel B shows the typical severity of the rough - eye phenotype . Data information : Representative phenotypes are shown in all panels . Scale bars , 100 μm .

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(A, B) Flies form well-organized compound eyes when they carry GMR-GAL4 without a target gene (A) but develop rough eyes when GMR-GAL4 drives expression of UAS-Foxn1 (B). Panel B shows the typical severity of the rough-eye phenotype. Data information: Representative phenotypes are shown in all panels. Scale bars, 100 μm.


( C , D ) Bristles are pigmented and properly shaped when flies carry hs - GAL4 without a target gene ( C ) but are often white , bent , and shortened when hs - GAL4 drives expression of UAS - Foxn1 ( D ) . Flies in panels C and D were raised at 25 ° C . Arrows denote examples of white bristles . Data information : Representative phenotypes are shown in all panels . Scale bars , 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Bristles are pigmented and properly shaped when flies carry hs-GAL4 without a target gene (C) but are often white, bent, and shortened when hs-GAL4 drives expression of UAS-Foxn1 (D). Flies in panels C and D were raised at 25°C. Arrows denote examples of white bristles. Data information: Representative phenotypes are shown in all panels. Scale bars, 100 μm.


AFF4 ( green ) and FOXN1 ( red ) are shown by immunofluorescence in the following wild - type tissue samples : ( B ) murine hair follicles , P9 ; DNA is stained by Hoechst dye 33258 ( blue ) . Co - localization of AFF4 and FOXN1 generates yellow color , visible in cell nuclei of all samples . Arrowheads mark the CH , cortex of the hair ; Scale bars , 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (B) murine hair follicles, P9; DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. Arrowheads mark the CH, cortex of the hair; Scale bars, 20 µm.


AFF4 ( green ) and FOXN1 ( red ) are shown by immunofluorescence in the following wild - type tissue samples : ( C ) developing murine epidermis , E16 . 5 ; ( D ) human epidermis , adult ; ( E ) murine thymus , P0 , outer region . DNA is stained by Hoechst dye 33258 ( blue ) . Co - localization of AFF4 and FOXN1 generates yellow color , visible in cell nuclei of all samples . In panel C , the fluorescent signal in the periderm ( P ) is nonspecific , as it is present when primary antibodies are omitted from the staining . Inner regions of the murine thymus ( not shown ) yielded the same extensive overlap of FOXN1 and AFF4 staining as that shown in panel E . Arrowheads mark the dermal / epidermal border ( C , D ) . CT , cortex of the thymus . Scale bars , 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (C) developing murine epidermis, E16.5; (D) human epidermis, adult; (E) murine thymus, P0, outer region. DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. In panel C, the fluorescent signal in the periderm (P) is nonspecific, as it is present when primary antibodies are omitted from the staining. Inner regions of the murine thymus (not shown) yielded the same extensive overlap of FOXN1 and AFF4 staining as that shown in panel E. Arrowheads mark the dermal/epidermal border (C, D). CT, cortex of the thymus. Scale bars, 20 µm.


Representative skin phenotypes of wild - type ( WT ) mice shown macroscopically The phenotypes compared are as follows : hair coats , 4 - month - old

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Representative skin phenotypes of wild-type (WT) mice shown macroscopically The phenotypes compared are as follows: hair coats, 4-month-old adults


Representative skin phenotypes of Foxn1nu / nu ( Nude ) , or KRT14 - cre ; Aff4flox / flox ( Aff4 cko ) mice are shown macroscopically or histologically after hematoxylin and eosin staining ( H - N ) . The phenotypes compared are as follows : hair coats , 4 - month - old adults ; ( E - G ) vibrissae , P7 ; ( H ) hair follicles with growing hairs , juveniles ; ( I - K ) regressing hair follicles , juveniles ; ( L - N ) epidermis , P9 . In panel H , micrographs from left to right are WT , Nude , and Aff4 cko . In panels I - K , HF marks examples of hair follicles . In panels L - M , arrowheads indicate the dermal / epidermal border ; arrows mark examples of bent hair shafts . Scale bars : H , L - N , 20 µm ; I - K , 40 µm .

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Representative skin phenotypes of Foxn1nu/nu (Nude), or KRT14-cre; Aff4flox/flox (Aff4 cko) mice are shown macroscopically or histologically after hematoxylin and eosin staining (H-N). The phenotypes compared are as follows: hair coats, 4-month-old adults; (E-G) vibrissae, P7; (H) hair follicles with growing hairs, juveniles; (I-K) regressing hair follicles, juveniles; (L-N) epidermis, P9. In panel H, micrographs from left to right are WT, Nude, and Aff4 cko. In panels I-K, HF marks examples of hair follicles. In panels L-M, arrowheads indicate the dermal/epidermal border; arrows mark examples of bent hair shafts. Scale bars: H, L-N, 20 µm; I-K, 40 µm.


( A - C ) Thymi from E18 . 5 embryos are shown : ( A ) intact with genotypes indicated , or ( B , C ) after sectioning , immunofluorescent staining for DLL4 ( red ) , and counterstaining of DNA ( blue ) . Sectioned samples are wild - type ( B ) or Aff4ko / ko ; Foxn1 + / - ( C ) . Scale bars : A , 1 mm ; B - C , 50 µm .

(A-C) Thymi from E18.5 embryos are shown: (A) intact with genotypes indicated, or (B, C) after sectioning, immunofluorescent staining for DLL4 (red), and counterstaining of DNA (blue). Sectioned samples are wild-type (B) or Aff4ko/ko; Foxn1+/- (C). Scale bars: A, 1 mm; B-C, 50 µm.


( C - E ) KRT86 ( red ) is shown by immunofluorescence in skin sections from wild - type ( WT ) , Foxn1 null , or Aff4 cko mice at P9 . DNA is stained blue . HS marks examples of hair shafts . Scale bar , 40 µm .

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(C-E) KRT86 (red) is shown by immunofluorescence in skin sections from wild-type (WT), Foxn1 null, or Aff4 cko mice at P9. DNA is stained blue. HS marks examples of hair shafts. Scale bar, 40 µm.


Graphs present pre - mRNA ( B , C ) assays of Krt86 using skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P6 ( B , C ) All values are derived from 3 independent experiments and presented as mean ± S . D . Pre - mRNA measurements were analyzed statistically using two - way ANOVA with post - hoc Sidak ' s multiple comparison test at a 95 % confidence interval .

Graphs present pre-mRNA (B, C) assays of Krt86 using skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P6 (B, C) All values are derived from 3 independent experiments and presented as mean ± S.D. Pre-mRNA measurements were analyzed statistically using two-way ANOVA with post-hoc Sidak's multiple comparison test at a 95% confidence interval.


Graphs present ChIP ( D - G ) assays of Krt86 using skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P7 ( D - G ) . ChIP assays were performed with antibodies to FOXN1 or AFF4 as indicated . All values are derived from 3 independent experiments and presented as mean ± S . D . ChIP assays were analyzed statistically using one - way ANOVA with post - hoc Tukey ' s multiple comparison test at a 95 % confidence interval . * * * * , p ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Graphs present ChIP (D-G) assays of Krt86 using skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P7 (D-G). ChIP assays were performed with antibodies to FOXN1 or AFF4 as indicated. All values are derived from 3 independent experiments and presented as mean ± S.D. ChIP assays were analyzed statistically using one-way ANOVA with post-hoc Tukey's multiple comparison test at a 95% confidence interval. ****, p ≤ 0.0001.


( B - G ) Graphs present ChIP assays of Krt86 using antibodies to total ( B , C ) , P - Ser5 ( D , E ) , or P - Ser2 ( F , G ) Pol II and skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P7 . All values are derived from at least 3 independent experiments and presented as mean ± S . D . Assay results were analyzed statistically using one - way ANOVA with post - hoc Tukey ' s multiple comparison test at a 95 % confidence interval . * * * * , p ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-G) Graphs present ChIP assays of Krt86 using antibodies to total (B, C), P-Ser5 (D, E), or P-Ser2 (F, G) Pol II and skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P7. All values are derived from at least 3 independent experiments and presented as mean ± S.D. Assay results were analyzed statistically using one-way ANOVA with post-hoc Tukey's multiple comparison test at a 95% confidence interval. ****, p ≤ 0.0001.


Keratinocytes were isolated from the skin of a 4 - year - old patient with severe - generalised RDEB linked to homozygous insertion - deletion in COL7A1 ( Hilal et al , 1993 ) . Cultured RDEB cells ( blue line ) were serially passaged for more than 4 months , displaying a growth potential similar to non - diseased control cells ( YF29 ) isolated from the foreskin of a newborn ( black line ) . To calculate the percentage of growing colonies , 100 to 1 , 000 cells were plated into indicator dishes at each passage . Cells were grown for 12 days , fixed and stained with rhodamine B . Colonies were scored as growing or aborted ( Barrandon Green , 1987 ) .

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Keratinocytes were isolated from the skin of a 4-year-old patient with severe-generalised RDEB linked to homozygous insertion-deletion in COL7A1 (Hilal et al, 1993). Cultured RDEB cells (blue line) were serially passaged for more than 4 months, displaying a growth potential similar to non-diseased control cells (YF29) isolated from the foreskin of a newborn (black line). To calculate the percentage of growing colonies, 100 to 1,000 cells were plated into indicator dishes at each passage. Cells were grown for 12 days, fixed and stained with rhodamine B. Colonies were scored as growing or aborted (Barrandon Green, 1987).


Clonal analysis demonstrated the presence of stem cells ( holoclones ) in a passage VII RDEB culture ( 95 % of growing colonies ) .

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Clonal analysis demonstrated the presence of stem cells (holoclones) in a passage VII RDEB culture (95% of growing colonies).


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . COLVII detection in clones by immunostaining . COLVII expression ( green ) was detectable in some clones ( 6 , 17 , 22 , 58 and 61 ) and not in others ( 3 , 24 and 54 ) ; nuclei were stained with Hoechst 33342 ( blue ) . Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3 - J2 feeder cells . Scale bar : 50 μm .

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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation.COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm.


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . Quantitative RT - PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes . All clones shown in ( A ) were transduced but expressed different levels of COL7A1 transcripts . Clones 6 , 17 , 22 , 54 , 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors ( YF29 and OR - CA , control 1 and 2 , respectively ) . The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1 .

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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Quantitative RT-PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1.


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . Determination of proviral rearrangements in transduced clones . A Southern blot was performed using genomic DNA of RDEB cells , clones and the infected mass culture from which the clones were isolated . Genomic DNA was digested with EcoRV and SpeI that cut at the 3 ′ and 5 ′ end of the provirus ( Supplementary Fig S2 ) and hybridised with a 907 - bp COL7A1 probe radiolabelled with 32P isotope . The upper band corresponded to the endogenous signal . The retroviral producer line Flp293A - E1aColVII1 was used as a control for the digested 9 . 6 - kb provirus ( proviral signal ) . Smaller bands corresponded to rearranged proviruses marked with an asterisk .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk.


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . Identification of stem cells producing COLVII . Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant , while clone 54 and surprisingly clone 22 did not ( see A ) . RDEB cells were used as a negative control and healthy donor cells as a positive control . The secreted matrix metalloproteinase 2 ( MMP2 ) was used as a loading control .

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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.


Serial cultivation of transduced and untransduced holoclones demonstrated that the growth potential of the stem cells was not affected by the production of COLVII . Non - COLVII - producing holoclone 54 ( black lines ) and COLVII - producing holoclone 6 ( red lines ) were serially transferred once a week until exhaustion ( Rochat et al , 1994 ) . Theoretical number of epidermal cells available for characterisation and CEA production from corrected ( clone 6 ) and uncorrected stem cells ( clone 54 ) calculated from the day of cloning . The colony - forming efficiency and the percentage of growing colonies for each passage were used to calculate the population doubling , the generation number and the total progeny of isolated stem cells . Both corrected and uncorrected epidermal stem cells show high growth potential in vitro .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Serial cultivation of transduced and untransduced holoclones demonstrated that the growth potential of the stem cells was not affected by the production of COLVII. Non-COLVII-producing holoclone 54 (black lines) and COLVII-producing holoclone 6 (red lines) were serially transferred once a week until exhaustion (Rochat et al, 1994).Theoretical number of epidermal cells available for characterisation and CEA production from corrected (clone 6) and uncorrected stem cells (clone 54) calculated from the day of cloning. The colony-forming efficiency and the percentage of growing colonies for each passage were used to calculate the population doubling, the generation number and the total progeny of isolated stem cells. Both corrected and uncorrected epidermal stem cells show high growth potential in vitro.


Immunodeficient SCID mice were transplanted with untransduced cultured RDEB keratinocytes ( left ) or the COLVII - secreting holoclone ( clone 6 ) ( right ) . Punch biopsies were obtained at various times post - transplantation ( PT ) , stained with haematoxylin / eosine ( H / E ) , for human leucocyte antigen - 1 ( HLA - 1 ) ( green ) and human COLVII ( red ) . RDEB keratinocytes generated a HLA - 1 - positive epidermis that adhered poorly to the dermo - epidermal junction ( DEJ ) ( arrow indicates a blister ) and absence of COLVII ( dotted line delimits the dermis ) , whereas the corrected keratinocytes produced an epidermis that adhered to the dermis and deposited COLVII ( red ) at the DEJ . Note the presence of KI67 - positive keratinocytes ( red ) in the basal and suprabasal layers in the corrected epidermis at 385 days post - transplantation , indicating that transplanted cells had self - renewed for more than a year . Scale bar : 50 μm .

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Immunodeficient SCID mice were transplanted with untransduced cultured RDEB keratinocytes (left) or the COLVII-secreting holoclone (clone 6) (right). Punch biopsies were obtained at various times post-transplantation (PT), stained with haematoxylin/eosine (H/E), for human leucocyte antigen-1 (HLA-1) (green) and human COLVII (red). RDEB keratinocytes generated a HLA-1-positive epidermis that adhered poorly to the dermo-epidermal junction (DEJ) (arrow indicates a blister) and absence of COLVII (dotted line delimits the dermis), whereas the corrected keratinocytes produced an epidermis that adhered to the dermis and deposited COLVII (red) at the DEJ. Note the presence of KI67-positive keratinocytes (red) in the basal and suprabasal layers in the corrected epidermis at 385 days post-transplantation, indicating that transplanted cells had self-renewed for more than a year. Scale bar: 50 μm.


Transmission electron microscopy ( TEM ) demonstrated the presence of anchoring fibrils ( arrows ) at the DEJ of the corrected epidermis ( middle right panel ) and in a normal human skin biopsy ( left panel ) . Note the absence of anchoring fibrils in the RDEB epidermis ( middle left panel ) . Detection of human COLVII by immunogold staining in the DEJ of corrected epidermis ( right panel ) . Scale bar : 250 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Transmission electron microscopy (TEM) demonstrated the presence of anchoring fibrils (arrows) at the DEJ of the corrected epidermis (middle right panel) and in a normal human skin biopsy (left panel). Note the absence of anchoring fibrils in the RDEB epidermis (middle left panel). Detection of human COLVII by immunogold staining in the DEJ of corrected epidermis (right panel). Scale bar: 250 nm.


Visualisation of COL7A1 sequence by FISH analysis in corrected stem cells at passage XII ( clone 6 ) . Five specific signals ( red ) were detected on five different chromosomes . As expected , a COL7A1 probe ( red ) hybridised to the two endogenous COL7A1 alleles located on the chromosomes 3 as identified by a specific centromeric probe ( Cen 3 ) and to three other chromosomes identified as chromosomes 2 , 11 and 22 by means of specific centromeric probes ( Cen 2 , Cen 11 and Cen 22 , respectively ) . The latter corresponded to proviruses . Determination of proviral integration sites in corrected stem cell . Genomic DNA from clone 6 was submitted to whole - genome sequencing together with PCR and subsequent Sanger sequencing to identify the integration sites to the base - pair level . Mate pairs that span from the viral sequence to a human chromosome were extracted and used to estimate integration regions . The reads were mapped to the hg19 reference sequence . Three integration sites were uncovered : one in chromosome 2 , one in chromosome 11 and one in chromosome 22 , and targeted genes were identified .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Visualisation of COL7A1 sequence by FISH analysis in corrected stem cells at passage XII (clone 6). Five specific signals (red) were detected on five different chromosomes. As expected, a COL7A1 probe (red) hybridised to the two endogenous COL7A1 alleles located on the chromosomes 3 as identified by a specific centromeric probe (Cen 3) and to three other chromosomes identified as chromosomes 2, 11 and 22 by means of specific centromeric probes (Cen 2, Cen 11 and Cen 22, respectively). The latter corresponded to proviruses.Determination of proviral integration sites in corrected stem cell. Genomic DNA from clone 6 was submitted to whole-genome sequencing together with PCR and subsequent Sanger sequencing to identify the integration sites to the base-pair level. Mate pairs that span from the viral sequence to a human chromosome were extracted and used to estimate integration regions. The reads were mapped to the hg19 reference sequence. Three integration sites were uncovered: one in chromosome 2, one in chromosome 11 and one in chromosome 22, and targeted genes were identified.


Analysis of the level of expression of targeted genes in corrected stem cells by quantitative RT - PCR . DARS was not significantly changed in clone 6 compared to the cells before transduction . Primers used annealed downstream of the proviral integration site . Error bars represent the standard deviation of three replicates . Identification of sequence abnormalities in the two alleles of the DARS - targeted gene in clone 6 by NGS . Reads were mapped to the hg19 reference sequence . Small insertion - deletions and SNP calling were performed and compared to GWAS databases . The mapping highlighted two indels and one SNP in the DARS genomic sequence . The indels were not associated with disease and the SNP was synonymous ( CCU - CCG both codons correspond to proline ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of the level of expression of targeted genes in corrected stem cells by quantitative RT-PCR. DARS was not significantly changed in clone 6 compared to the cells before transduction. Primers used annealed downstream of the proviral integration site. Error bars represent the standard deviation of three replicates.Identification of sequence abnormalities in the two alleles of the DARS-targeted gene in clone 6 by NGS. Reads were mapped to the hg19 reference sequence. Small insertion-deletions and SNP calling were performed and compared to GWAS databases. The mapping highlighted two indels and one SNP in the DARS genomic sequence. The indels were not associated with disease and the SNP was synonymous (CCU-CCG both codons correspond to proline).


Upper panel : expression of proteins associated with the acquisition of immortalisation process involved in senescence , in evasion of growth suppression and in apoptosis ( Hanahan Weinberg , 2011 ) . The level of P53 , P21 , RAS and P16 was similar in clone 6 , untransduced cells and mass culture from four independent infections , significantly different from the squamous cell carcinoma cell line SCC - 13 . Lower panel : the phosphorylation state of the PRB restriction point was maintained in clone 6 and untransduced recessive dystrophic epidermolysis bullosa ( RDEB ) cells , whereas PRB was heavily phosphorylated in transformed cells ( SCC - 13 ) . Appropriate loading controls were used for each cellular extract ( GAPDH for cytoplasmic extracts , histone H3 for nuclear extracts and tubulin for whole - cell extracts . H3 for histone H3 , ppRB for hyperphosphorylated pRB and pRB for hypophosphorylated PRB ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Upper panel: expression of proteins associated with the acquisition of immortalisation process involved in senescence, in evasion of growth suppression and in apoptosis (Hanahan Weinberg, 2011). The level of P53, P21, RAS and P16 was similar in clone 6, untransduced cells and mass culture from four independent infections, significantly different from the squamous cell carcinoma cell line SCC-13. Lower panel: the phosphorylation state of the PRB restriction point was maintained in clone 6 and untransduced recessive dystrophic epidermolysis bullosa (RDEB) cells, whereas PRB was heavily phosphorylated in transformed cells (SCC-13). Appropriate loading controls were used for each cellular extract (GAPDH for cytoplasmic extracts, histone H3 for nuclear extracts and tubulin for whole-cell extracts. H3 for histone H3, ppRB for hyperphosphorylated pRB and pRB for hypophosphorylated PRB).


Transduced clone 6 had a diploid karyotype at passage XVI ( see also Fig5A ) .

Transduced clone 6 had a diploid karyotype at passage XVI (see also Fig5A).


Transduced clones were not tumorigenic . The tumorigenic potential of the clones was tested by tumour formation in nude mice . Transduced RDEB holoclones ( blue ) [ clone 6 ( square , n = 4 ) passage X , clone 22 ( circle , n = 2 ) passage XI , clone 54 ( cross , n = 4 ) passage X ] were not tumorigenic when injected subcutaneously in athymic mice as were untransduced RDEB keratinocytes ( passage VII ) . SCC - 13 , a squamous cell carcinoma cell line ( black ) ( lozenge , n = 2 ) , was used as a positive control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Transduced clones were not tumorigenic. The tumorigenic potential of the clones was tested by tumour formation in nude mice. Transduced RDEB holoclones (blue) [clone 6 (square, n = 4) passage X, clone 22 (circle, n = 2) passage XI, clone 54 (cross, n = 4) passage X] were not tumorigenic when injected subcutaneously in athymic mice as were untransduced RDEB keratinocytes (passage VII). SCC-13, a squamous cell carcinoma cell line (black) (lozenge, n = 2), was used as a positive control.


To test whether the transduced RDEB keratinocytes had disseminated after the generation of an epidermis onto SCID mice ( see Fig4C ) , the internal organs of the recipient mice ( 3 mice for clone 6 and 2 mice for clone 22 ) were harvested 385 days post - transplantation and analysed for COL7A1 proviral sequences . No COL7A1 sequence was detected . A mouse transplanted with a holoclone obtained from a healthy donor ( YF29 ) was used as an internal control . PCR - positive controls were genomic DNA from transduced cells ( holoclone 6 ) and cDNAs isolated from healthy keratinocytes . B - actin was run as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

To test whether the transduced RDEB keratinocytes had disseminated after the generation of an epidermis onto SCID mice (see Fig4C), the internal organs of the recipient mice (3 mice for clone 6 and 2 mice for clone 22) were harvested 385 days post-transplantation and analysed for COL7A1 proviral sequences. No COL7A1 sequence was detected. A mouse transplanted with a holoclone obtained from a healthy donor (YF29) was used as an internal control. PCR-positive controls were genomic DNA from transduced cells (holoclone 6) and cDNAs isolated from healthy keratinocytes. B-actin was run as a loading control.


5C or mES medium was used during reprogramming . On day 15 , Oct4GFP + colonies were counted ( A ) .

O O O O O O O O O O O O O O O O O O O O O O

5C or mES medium was used during reprogramming. On day 15, Oct4GFP+ colonies were counted (A).


5C or mES medium was used during reprogramming . The percentages of Oct4GFP + cells were also determined with FACS ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O

5C or mES medium was used during reprogramming. The percentages of Oct4GFP+ cells were also determined with FACS (B).


( C ) The expression of several core pluripotency markers was determined with qPCR at different time points during reprogramming and normalized against those in MEFs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The expression of several core pluripotency markers was determined with qPCR at different time points during reprogramming and normalized against those in MEFs.


( D - E ) Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument . Increase in extracellular acidification rate ( ECAR ) after adding glucose was considered as glycolysis ability of the cells ( D ) , while the decrease in oxygen consumption rate ( OCR ) was considered as the ATP production ability of the cells ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-E) Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument. Increase in extracellular acidification rate (ECAR) after adding glucose was considered as glycolysis ability of the cells (D), while the decrease in oxygen consumption rate (OCR) was considered as the ATP production ability of the cells (E).


( F - G ) Expression of Hif1α was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming ( F ) . The numbers of Oct4GFP + colonies were determined on day 15 ( G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F-G) Expression of Hif1α was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming (F). The numbers of Oct4GFP+ colonies were determined on day 15 (G).


( H - I ) Expression of Pdk1 / 2 was modulated with a retrovirus system , and oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) were used during reprogramming ( H ) . The numbers of Oct4GFP + colonies were determined on day 15 ( I ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H-I) Expression of Pdk1/2 was modulated with a retrovirus system, and oligomycin (1 µM) and 2-DG (5 mM) were used during reprogramming (H). The numbers of Oct4GFP+ colonies were determined on day 15 (I).


( A - B ) 5C and mES medium were used during reprogramming . Representative alkaline phosphatase ( AP ) staining on day 15 was provided in ( A ) . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) 5C and mES medium were used during reprogramming. Representative alkaline phosphatase (AP) staining on day 15 was provided in (A). AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (B).


( C ) Thirty - two epigenetic factors were selected because they had higher expression in 5C - Oct4GFP - than in mES - Oct4GFP - . Their expression in ESCs , iPSCs , and pre - iPSCs in two previous reported assay ( GSE14012 and GSE10871 ) was also listed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Thirty-two epigenetic factors were selected because they had higher expression in 5C-Oct4GFP- than in mES-Oct4GFP-. Their expression in ESCs, iPSCs, and pre-iPSCs in two previous reported assay (GSE14012 and GSE10871) was also listed.


( D ) The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , or Wdr5 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 .

(D) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15.


During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The expression of pluripotency markers was determined with qPCR on day 6 during reprogramming ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The expression of pluripotency markers was determined with qPCR on day 6 during reprogramming (E).


During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The numbers of Oct4GFP + colonies were determined on day 15 ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The numbers of Oct4GFP+ colonies were determined on day 15 (F).


During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The histone methylation on core pluripotency loci was determined on day 6 with ChIP - qPCR ( G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The histone methylation on core pluripotency loci was determined on day 6 with ChIP-qPCR (G).


( A ) The enrichment of HIF1α , SNAI2 , TWIST1 / 2 , and ZEB1 binding sites on the promoters of Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 was determined by Pscan Zambelli et al , 2009 ( ) . The resulted Z - scores were listed . " Max . M " suggested the maximum Z - score generated with SNAI2 , TWIST1 / 2 , and ZEB1 binding sites on one indicated promoter . " Average " suggested the average Z - score of all transcriptional factors tested in the Pscan software on one indicated promoter .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The enrichment of HIF1α, SNAI2, TWIST1/2, and ZEB1 binding sites on the promoters of Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 was determined by Pscan Zambelli et al, 2009(). The resulted Z-scores were listed. "Max. M" suggested the maximum Z-score generated with SNAI2, TWIST1/2, and ZEB1 binding sites on one indicated promoter. "Average" suggested the average Z-score of all transcriptional factors tested in the Pscan software on one indicated promoter.


The expression of H1f1α , Snai2 , Twist1 / 2 , and Zeb1 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming with mES ( B ) The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs . The comparisons were performed between all groups and corresponding Flag groups .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The expression of H1f1α, Snai2, Twist1/2, and Zeb1 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming with mES (B) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs. The comparisons were performed between all groups and corresponding Flag groups.


The expression of H1f1α , Snai2 , Twist1 / 2 , and Zeb1 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming with 5C medium ( C ) . The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs . The comparisons were performed between all groups and corresponding Flag groups .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The expression of H1f1α, Snai2, Twist1/2, and Zeb1 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming with 5C medium (C). The expression of Bmi1, Ctcf, Ezh2, Kdm2b, Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs. The comparisons were performed between all groups and corresponding Flag groups.


pre - iPSCs were isolated during reprogramming with mES - Vc , mES or 5C medium . These pre - iPSCs were further cultured with a different medium ( mES - Vc and mES medium ) for seven days . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . The numbers of Oct4GFP + colonies were determined on day 7 ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

pre-iPSCs were isolated during reprogramming with mES-Vc, mES or 5C medium. These pre-iPSCs were further cultured with a different medium (mES-Vc and mES medium) for seven days. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. The numbers of Oct4GFP+ colonies were determined on day 7 (E).


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 ( G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (G).


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . In addition , energy metabolism was determined on day 6 with Seahorse

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. In addition, energy metabolism was determined on day 6 with Seahorse instrument


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . Cell migration was determined by measuring the migration rate with live - cell imaging on day 6 ( J ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. Cell migration was determined by measuring the migration rate with live-cell imaging on day 6 (J).


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , or Wdr5 was determined on day 6 with qPCR ( K ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was determined on day 6 with qPCR (K).


Expression of Hif1α was modulated via a retrovirus system or small - molecule compounds , oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) , during reprogramming . The expression of mesenchymal markers ( A )

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

Expression of Hif1α was modulated via a retrovirus system or small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM), during reprogramming. The expression of mesenchymal markers (A)


Expression of Hif1α was modulated via a retrovirus system or small - molecule compounds , oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) , during reprogramming . The expression of mesenchymal markers cell migration ( B ) was determined with qPCR and live - cell imaging , respectively , on day 6 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of Hif1α was modulated via a retrovirus system or small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM), during reprogramming. The expression of mesenchymal markers cell migration (B) was determined with qPCR and live-cell imaging, respectively, on day 6.


Repsox ( 1 μM ) was used on day 2 - 7 to inhibit early EMT during reprogramming with 5C medium . Except for OK + M + S group , the four Yamanaka factors were delivered simultaneously . Energy metabolism was determined on day 6 with Seahorse instrument ( E - F ) .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Repsox (1 μM) was used on day 2-7 to inhibit early EMT during reprogramming with 5C medium. Except for OK+M+S group, the four Yamanaka factors were delivered simultaneously. Energy metabolism was determined on day 6 with Seahorse instrument (E-F).


Repsox ( 1 μM ) was used on day 2 - 7 to inhibit early EMT during reprogramming with 5C medium . The expression of glycolysis markers was determined on day 6 with qPCR ( G ) .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Repsox (1 μM) was used on day 2-7 to inhibit early EMT during reprogramming with 5C medium. The expression of glycolysis markers was determined on day 6 with qPCR (G).


On day 15 during reprogramming , the expression of E - cadherin ( E - CAD ) and N - cadherin ( N - CAD ) , was determined with immunofluorescence together with the morphology in Oct4GFP + colonies ( A , Scale bar , 25 μM ) .

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On day 15 during reprogramming, the expression of E-cadherin (E-CAD) and N-cadherin (N-CAD), was determined with immunofluorescence together with the morphology in Oct4GFP+ colonies (A, Scale bar, 25 μM).


After sorting Oct4GFP + cells out ( 5C - Oct4GFP + and mES - Oct4GFP + cells ) , the expression of markers of different pluripotent states ( B )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells), the expression of markers of different pluripotent states (B)


After sorting Oct4GFP + cells out ( 5C - Oct4GFP + and mES - Oct4GFP + cells ) , the expression of markers of Xist ( C )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells), the expression of markers of Xist (C)


After sorting Oct4GFP + cells out ( 5C - Oct4GFP + and mES - Oct4GFP + cells ) their abilities to form chimeras ( D ) were determined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells) their abilities to form chimeras (D) were determined.


( F ) The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days , and the morphology changed towards R1 ESCs ( naïve PSCs ) and R1 ESCs - primed ( primed PSCs ) , respectively . The obtained cells were maintained for additional 20 passages . The 5C - Oct4GFP + cells cultured in primed medium lost GFP fluorescence . R1 ESCs - primed were primed PSCs generated from R1 ESCs . Scale bar , 100 μM .

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(F) The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days, and the morphology changed towards R1 ESCs (naïve PSCs) and R1 ESCs-primed (primed PSCs), respectively. The obtained cells were maintained for additional 20 passages. The 5C-Oct4GFP+ cells cultured in primed medium lost GFP fluorescence. R1 ESCs-primed were primed PSCs generated from R1 ESCs. Scale bar, 100 μM.


The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days , and the morphology changed towards R1 ESCs ( naïve PSCs ) and R1 ESCs - primed ( primed PSCs ) , respectively . ( G ) PSCs mentioned in ( F ) were used for RNA - Seq analysis . Similarity among gene expression profiles was determined .

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The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days, and the morphology changed towards R1 ESCs (naïve PSCs) and R1 ESCs-primed (primed PSCs), respectively. (G) PSCs mentioned in (F) were used for RNA-Seq analysis. Similarity among gene expression profiles was determined.


The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days to generate 5C - Oct4GFP + ( naïve ) and 5C - Oct4GFP + ( primed ) cells . The expression of Xist ( H )

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The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days to generate 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) cells. The expression of Xist (H)


The isolated 5C - Oct4GFP + cells were cultured in naïve or primed medium for three days to generate 5C - Oct4GFP + ( naïve ) and 5C - Oct4GFP + ( primed ) cells . the abilities to form chimeras ( I - J ) were determined .

The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days to generate 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) cells. the abilities to form chimeras (I-J) were determined.


The expression Oct4GFP and E - cadherin was determined with FACS on day 15 during reprogramming ( A ) .

The expression Oct4GFP and E-cadherin was determined with FACS on day 15 during reprogramming (A).


Based on the FACS results , the expression of Oct4GFP and E - cadherin was summarized in Oct4GFP + E - cadherin + cells ( B ) and in Oct4GFP - E - cadherin - cells ( C ) .

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Based on the FACS results, the expression of Oct4GFP and E-cadherin was summarized in Oct4GFP+E-cadherin+ cells (B) and in Oct4GFP-E-cadherin- cells (C).


The percentage of E - cadherin + cells in Oct4GFP + cells was listed in ( D ) , while the percentage of Oct4GFP + cells in E - cadherin + cells was listed in ( E ) .

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The percentage of E-cadherin+ cells in Oct4GFP+ cells was listed in (D), while the percentage of Oct4GFP+ cells in E-cadherin+ cells was listed in (E).


( F - G ) Energy metabolism was analyzed in 5C - Oct4GFP + and mES - Oct4GFP + cells with Seahorse instrument .

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(F-G) Energy metabolism was analyzed in 5C-Oct4GFP+ and mES-Oct4GFP+ cells with Seahorse instrument.


The numbers of three types of Oct4GFP + colonies ( J )

The numbers of three types of Oct4GFP+ colonies (J)


the expression of four epithelial markers in Oct4GFP + cells ( K ) were determined on day 15 . " Average " in ( K ) suggested the average expression of four epithelia markers .

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the expression of four epithelial markers in Oct4GFP+ cells (K) were determined on day 15. "Average" in (K) suggested the average expression of four epithelia markers.


( A - B ) Energy metabolism was analyzed with 5C - Oct4GFP + , R1 ESCs ( naïve PSCs ) , and R1 ESCs - primed ( primed PSCs ) with Seahorse instrument .

(A-B) Energy metabolism was analyzed with 5C-Oct4GFP+, R1 ESCs (naïve PSCs), and R1 ESCs-primed (primed PSCs) with Seahorse instrument.


( C ) Expression of epithelial , mesenchymal , glycolysis and oxidative phosphorylation ( OXPHOS ) markers ( listed in Dataset EV4 ) was summarized in R1 ESCs , 5C - Oct4GFP + ( naïve ) , 5C - Oct4GFP + , 5C - Oct4GFP + ( primed ) , and R1 ESCs - primed , based on the current RNA - Seq . For each marker , Log2 values of FKPM were normalized by subtracting the average of five groups . Log2 values of FKPM of each group of markers were then averaged and plotted . 5C - Oct4GFP + ( naïve ) and 5C - Oct4GFP + ( primed ) were 5C - Oct4GFP + cultured in naïve and primed medium , respectively , for three days .

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(C) Expression of epithelial, mesenchymal, glycolysis and oxidative phosphorylation (OXPHOS) markers (listed in Dataset EV4) was summarized in R1 ESCs, 5C-Oct4GFP+ (naïve), 5C-Oct4GFP+, 5C-Oct4GFP+ (primed), and R1 ESCs-primed, based on the current RNA-Seq. For each marker, Log2 values of FKPM were normalized by subtracting the average of five groups. Log2 values of FKPM of each group of markers were then averaged and plotted. 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) were 5C-Oct4GFP+ cultured in naïve and primed medium, respectively, for three days.


( D ) Genes with higher expression at different developmental stages were selected out from previously reported RNA - seq ( Data ref : Bertone , 2015 ) . " Mor " , " ICM " , " pre - EPI " and " post - EPI " represented genes that have high expression only in morula , inner cell mass , pre - implantation epiblast and post - implantation epiblast , respectively . " Mor + ICM " , " ICM + pre " , and " EPI " indicated genes that have high expression in both morula and inner cell mass , in both inner cell mass and pre - implantation epiblast , and in both pre - implantation and post - implantation epiblast , respectively . The results of previously reported RNA - seq were summarized in the left panel , while the expression of these genes in current RNA - seq was summarized in the right panel . Log2 values of FKPM were presented similarly as in ( C ) .

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(D) Genes with higher expression at different developmental stages were selected out from previously reported RNA-seq (Data ref: Bertone, 2015). "Mor", "ICM", "pre-EPI" and "post-EPI" represented genes that have high expression only in morula, inner cell mass, pre-implantation epiblast and post-implantation epiblast, respectively. "Mor+ICM", "ICM+pre", and "EPI" indicated genes that have high expression in both morula and inner cell mass, in both inner cell mass and pre-implantation epiblast, and in both pre-implantation and post-implantation epiblast, respectively. The results of previously reported RNA-seq were summarized in the left panel, while the expression of these genes in current RNA-seq was summarized in the right panel. Log2 values of FKPM were presented similarly as in (C).


( F ) Markers of 5C , naïve and primed states ( Dataset EV4 ) were used to analyze expression profiles of epiblast during the early development of mouse embryos ( Data ref : Mohammed et al , 2017a ) ( F ) . Single - cell was plotted based on the average expression of each group of markers .

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(F) Markers of 5C, naïve and primed states (Dataset EV4) were used to analyze expression profiles of epiblast during the early development of mouse embryos (Data ref: Mohammed et al, 2017a) (F). Single-cell was plotted based on the average expression of each group of markers.


( G ) The average expression of both naïve and primed markers was calculated based on GSE100597 . The top - 2 cells from E4 . 5 epiblast and top - 5 cells from E5 . 5 epiblast were selected . The expression of several naïve markers ( Dppa3 , Esrrb , Klf4 , Klf5 , Tbx3 , and Zfp42 ) and several mesenchymal markers ( Fgf5 , Lef1 , Nodal , and Pou3f1 ) in these selected cells were listed ( upper part ) . The average expression of these genes in E3 . 5 , 4 . 5 , 5 . 5 and 6 . 5 epiblast were also listed ( lower part ) . These results were all normalized against the average expression of these genes in all tested cells .

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(G) The average expression of both naïve and primed markers was calculated based on GSE100597. The top-2 cells from E4.5 epiblast and top-5 cells from E5.5 epiblast were selected. The expression of several naïve markers (Dppa3, Esrrb, Klf4, Klf5, Tbx3, and Zfp42) and several mesenchymal markers (Fgf5, Lef1, Nodal, and Pou3f1) in these selected cells were listed (upper part). The average expression of these genes in E3.5, 4.5, 5.5 and 6.5 epiblast were also listed (lower part). These results were all normalized against the average expression of these genes in all tested cells.


( H ) The expression of representative markers of 5C , naïve and primed states was summarized in E3 . 5 , 4 . 5 , 5 . 5 and 6 . 5 epiblast .

(H) The expression of representative markers of 5C, naïve and primed states was summarized in E3.5, 4.5, 5.5 and 6.5 epiblast.


Cells from E5 . 5 epiblast were separated into 8 groups ( I - VIII ) basing on the average expression of epithelial , mesenchymal , and glycolysis markers ( H ) . The average expression of three groups of markers were summarized in ( J ) .

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Cells from E5.5 epiblast were separated into 8 groups (I-VIII) basing on the average expression of epithelial, mesenchymal, and glycolysis markers (H). The average expression of three groups of markers were summarized in (J).


The expression of six important markers of 5C state was summarized in these eight groups of cells ( K )

The expression of six important markers of 5C state was summarized in these eight groups of cells (K)


b ) Box plot showing the distribution of log2 ( FC ) of all or selected miR - 29a targets in the Data ref : Mazziotti et al , 2017 dataset of age - regulated genes in the visual cortex . Notches represent confidence intervals for the median . Whiskers indicate 95th and 5th percentile , the box marks the 25th and 75th percentile . Significance vs . all detected genes was assessed by Wilcoxon ' s test ( miR - 29a targets p = 4 . 9x10 - 14 , top 200 miR - 29a targets p = 3 . 7x10 - 38 ) .

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b) Box plot showing the distribution of log2(FC) of all or selected miR-29a targets in the Data ref: Mazziotti et al, 2017 dataset of age-regulated genes in the visual cortex. Notches represent confidence intervals for the median. Whiskers indicate 95th and 5th percentile, the box marks the 25th and 75th percentile. Significance vs. all detected genes was assessed by Wilcoxon's test (miR-29a targets p=4.9x10-14, top 200 miR-29a targets p=3.7x10-38).


( d , e ) Mature miR - 29a ( n = 3 / 4 mice for each age ; one - way ANOVA , p < 0 . 0001 ; post hoc Tukey ' s test * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns not significant ) and Dnmt3a expression level ( normalized to P10 ) at different ages measured by qPCR ( n = 3 / 4 mice for each age ; one - way ANOVA , p < 0 . 0001 ; post hoc Tukey ' s test : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns not significant ) . One way ANOVA p - values are reported . Error bars represent ± SEM . f ) Scatter plot showing correlation of miR - 29a and Dnmt3a levels in the visual cortex display a strong negative correlation ( Pearson correlation : r = - 0 . 817 , p < 0 . 001 ) . Each circle represents data from a single animal .

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(d,e) Mature miR-29a (n=3/4 mice for each age; one-way ANOVA, p<0.0001; post hoc Tukey's test ***p<0.001, ****p<0.0001, ns not significant) and Dnmt3a expression level (normalized to P10) at different ages measured by qPCR (n=3/4 mice for each age; one-way ANOVA, p<0.0001; post hoc Tukey's test: *p<0.05, **p<0.01, ****p<0.0001, ns not significant). One way ANOVA p-values are reported. Error bars represent ±SEM. f) Scatter plot showing correlation of miR-29a and Dnmt3a levels in the visual cortex display a strong negative correlation (Pearson correlation: r=-0.817, p<0.001). Each circle represents data from a single animal.


g ) Representative examples of in situ hybridization on visual cortex ( left P25 , right P120 ) using a LNA probe against miR - 29a . Scale bar 180 µm . Cortical layers are delimited by roman numerals and

g) Representative examples of in situ hybridization on visual cortex (left P25, right P120) using a LNA probe against miR-29a. Scale bar 180 µm. Cortical layers are delimited by roman numerals and brackets


h ) Example of co - staining for PV ( green ) , miR - 29a ( red ) and nuclear marker ( blue ) in a P120 mouse . White triangles represent PV + / miR - 29a + cells . Scale bar 20 µm .

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h) Example of co-staining for PV (green), miR-29a (red) and nuclear marker (blue) in a P120 mouse. White triangles represent PV+/miR-29a+ cells. Scale bar 20 µm.


Mature miR - 29a expression ( miR29 ) in visually deprived mice : a ) normal rearing ( NR , N = 5 ) vs dark rearing ( DRb , N = 5 ) ; t - test , p = 0 . 19 , b ) NR ( N = 4 ) vs dark rearing from P10 ( DRp10 , N = 5 ; t - test , p = 0 . 70 . In both experiments , analysis was performed at

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Mature miR-29a expression (miR29) in visually deprived mice: a) normal rearing (NR, N=5) vs dark rearing (DRb, N=5); t-test, p=0.19, b) NR (N=4) vs dark rearing from P10 (DRp10, N=5; t-test, p=0.70. In both experiments, analysis was performed at P25


Mature miR - 29a expression ( miR29 ) in visually deprived mice : c ) one week period of dark rearing beginning at P30 ( DRw ) followed by a 4 hour exposure to light ( DRw + 4h light ) vs control animals ( ctr , N = 4 for each group ; one - way ANOVA , p = 0 . 68 )

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Mature miR-29a expression (miR29) in visually deprived mice: c) one week period of dark rearing beginning at P30 (DRw) followed by a 4 hour exposure to light (DRw+4h light) vs control animals (ctr, N=4 for each group; one-way ANOVA, p=0.68)


Mature miR - 29a expression ( miR29 ) in visually deprived mice : d ) days of MD beginning at P25 , contralateral cortex vs ipsilateral cortex to deprived eye , N = 5 ; paired t test , p = 0 . 52 ) . Error bars represent ± SEM , symbols represent individual cases .

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Mature miR-29a expression (miR29) in visually deprived mice: d) days of MD beginning at P25, contralateral cortex vs ipsilateral cortex to deprived eye, N=5; paired t test, p=0.52). Error bars represent ±SEM, symbols represent individual cases.


b ) MiR - 29a and Dnmt3a expression in the cortex treated with scrambled or miR - 29a mimic ( N = 3 mice ) . Data were normalized to the untreated cortex of the same animal . Mir - 29a : scr vs mim29a , t - test , p = 0 . 005 ; Dnmt3a scr vs mim29a t - test , p < 0 . 0001 .

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b) MiR-29a and Dnmt3a expression in the cortex treated with scrambled or miR-29a mimic (N=3 mice). Data were normalized to the untreated cortex of the same animal. Mir-29a: scr vs mim29a, t-test, p=0.005; Dnmt3a scr vs mim29a t-test, p<0.0001.


c ) IOS analysis . Ocular dominance index ( ODI ) of deprived animals treated with scrambled ( scr , N = 6 black lines ) or miR - 29a mimic ( mimic29 , N = 8 orange lines ) , or left untreated ( MD no treat , N = 5 grey lines ) before and after 3 days of MD . Dashed lines represent single animals , continuous lines the group average . two - way ANOVA RM , treatment X time , p = 0 . 028 ; post hoc Sidak ' s comparison , post - MD mim29a vs scr p < 0 . 05 , mim29a vs MD no treat p < 0 . 01 ; pre - MD scr vs post - MD scr p < 0 . 001 ; pre - MD MD no treat vs post - MD MD no treat p < 0 . 01 ; other comparisons not significant ) .

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c) IOS analysis. Ocular dominance index (ODI) of deprived animals treated with scrambled (scr, N=6 black lines) or miR-29a mimic (mimic29, N=8 orange lines), or left untreated (MD no treat, N=5 grey lines) before and after 3 days of MD. Dashed lines represent single animals, continuous lines the group average. two-way ANOVA RM, treatment X time, p= 0.028; post hoc Sidak's comparison, post-MD mim29a vs scr p<0.05, mim29a vs MD no treat p<0.01; pre-MD scr vs post-MD scr p<0.001; pre-MD MD no treat vs post-MD MD no treat p<0.01; other comparisons not significant).


d ) The effects of MD on expression of Npas IV and Bdnf exIV is reported as ratio between expression level of deprived and nondeprived cortex for animals treated with scrambled ( scr + MD ) and miR - 29a mimic ( mim29a + MD ) . Each symbol represents the result of a single mouse . ( MD + scr vs MD + mim29a ; N = 4 ; t - test , Npas4 : p = 0 . 038 ; Bdnf exIV : p = 0 . 049 ) .

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d) The effects of MD on expression of Npas IV and Bdnf exIV is reported as ratio between expression level of deprived and nondeprived cortex for animals treated with scrambled (scr+MD) and miR-29a mimic (mim29a+MD). Each symbol represents the result of a single mouse. (MD+scr vs MD+mim29a; N=4; t-test, Npas4: p=0.038; Bdnf exIV: p=0.049).


b ) Effects of amiR - 29a on the expression of miR - 29a and Dnmt3a . Fold change values with respect to the contralateral untreated cortex are reported ( ± SEM ) for mice treated with amiR - 29a or scr . scr vs amiR - 29a ; N = 3 t - test , miR - 29a * * p = 0 . 0010 ; Dnmt3a * * * * p = 0 . 0019 .

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b) Effects of amiR-29a on the expression of miR-29a and Dnmt3a. Fold change values with respect to the contralateral untreated cortex are reported (±SEM) for mice treated with amiR-29a or scr. scr vs amiR-29a; N=3 t-test, miR-29a **p=0.0010; Dnmt3a ****p=0.0019.


c ) Distribution of log2 ( FC ) for the significant cases in transcriptomics ( N = 4 scr treated and N = 3 amiR - 29a treated biological replicates ) and proteomics ( N = 4 biological replicates per group ) datasets of animals treated with amiR - 29a versus scr animals . The black line represents the average . ( d , e ) Box plot showing distribution of log2 ( FC ) of miR29a targets as d ) transcripts ( RNA ) and e ) proteins ( PROT ) in transcriptomic and proteomic datasets of adult animals treated with amiR - 29a . Notches correspond to confidence intervals of the median . Whiskers indicate 95th and 5th percentile , the box marks the 25th and 75th percentile . Significance vs . all detected genes and proteins , respectively , assessed by Wilcoxon ' s test ( p - values shown in figure ) .

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c) Distribution of log2(FC) for the significant cases in transcriptomics (N=4 scr treated and N=3 amiR-29a treated biological replicates) and proteomics (N=4 biological replicates per group) datasets of animals treated with amiR-29a versus scr animals. The black line represents the average. (d,e) Box plot showing distribution of log2(FC) of miR29a targets as d) transcripts (RNA) and e) proteins (PROT) in transcriptomic and proteomic datasets of adult animals treated with amiR-29a. Notches correspond to confidence intervals of the median. Whiskers indicate 95th and 5th percentile, the box marks the 25th and 75th percentile. Significance vs. all detected genes and proteins, respectively, assessed by Wilcoxon's test (p-values shown in figure).


f ) Box plots reporting distribution of log2 ( FC ) of age down - regulated miR29a targets after amiR - 29a treatment ( N = 4 scr treated and N = 3 amiR - 29a treated biological replicates ) . Notches correspond to confidence intervals of the median . Whiskers indicate 95th and 5th percentile , the box marks the 25th and 75th percentile . Significance was assessed with Wilcoxon ' s test ( p - values shown in figure ) .

f) Box plots reporting distribution of log2(FC) of age down-regulated miR29a targets after amiR-29a treatment (N=4 scr treated and N=3 amiR-29a treated biological replicates). Notches correspond to confidence intervals of the median. Whiskers indicate 95th and 5th percentile, the box marks the 25th and 75th percentile. Significance was assessed with Wilcoxon's test (p-values shown in figure).


h ) Scatter plot correlating the expression changes of genes significantly regulated by age ( log2FC , x - axis ) and miR - 29a antagonization by amiR29a in adult mice ( log2FC , y - axis ) . ( Spearman r = - 0 . 22 p = 1 . 4x10 - 8 ; Fisher test showing significant enrichment in the IV quadrant p = 2 . 2x10 - 16 ) . The number of genes for each quadrant is reported in figure .

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h) Scatter plot correlating the expression changes of genes significantly regulated by age (log2FC, x-axis) and miR-29a antagonization by amiR29a in adult mice (log2FC, y-axis). (Spearman r=-0.22 p=1.4x10-8; Fisher test showing significant enrichment in the IV quadrant p=2.2x10-16). The number of genes for each quadrant is reported in figure.


i ) Scatter plot correlating the expression changes induced by miR - 29a antagonization in adult mice at the transcript - ( log2FC , x - axis ) and protein - level ( log2FC , y - axis ) . Spearman coefficient correlation , r = 0 . 11 ; p = 0 . 006 , Fisher test showing significant enrichment in the I quadrant p = 0 . 03 . The number of genes for each quadrant is reported in figure .

i) Scatter plot correlating the expression changes induced by miR-29a antagonization in adult mice at the transcript- (log2FC, x-axis) and protein-level (log2FC, y-axis). Spearman coefficient correlation, r=0.11; p=0.006, Fisher test showing significant enrichment in the I quadrant p=0.03. The number of genes for each quadrant is reported in figure.


a ) VEP analysis . Ratio between contralateral and ipsilateral eye responses ( C / I ratio ± SEM ) in animals treated with saline and not deprived ( sal no MD ) , with scrambled LNA and MD ( scr MD ) , with amiR - 29a LNA and MD ( amiR - 29a MD ) . N = 6 / 7 ; one - way ANOVA , p = 0 . 0003 ; post hoc Tukey ' s test : sal noMD vs scr + MD , ns ; sal noMD vs amiR - 29a + MD , p = 0 . 0002 ; scr + MD vs amiR - 29a + MD , p = 0 . 0169 .

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a) VEP analysis. Ratio between contralateral and ipsilateral eye responses (C/I ratio ± SEM) in animals treated with saline and not deprived (sal no MD), with scrambled LNA and MD (scr MD), with amiR-29a LNA and MD (amiR-29a MD). N=6/7; one-way ANOVA, p=0.0003; post hoc Tukey's test: sal noMD vs scr+MD, ns; sal noMD vs amiR-29a+MD, p=0.0002; scr+MD vs amiR-29a+MD, p=0.0169.


b ) IOS analysis . ODI ± SEM of deprived animals treated with scr ( N = 5 , black lines ) and amiR - 29a LNA ( N = 6 , blue lines ) . Dashed lines connect pre - and post - MD ODI values of each animal . Solid lines represent the group average . Two - way ANOVA RM , treatment X time , p = 0 . 0074 ; post hoc Sidak ' s test , pre - MD scr vs post - MD scr , ns ; pre - MD vs post - MD amiR - 29a , p = 0 . 0005 .

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b) IOS analysis. ODI ± SEM of deprived animals treated with scr (N=5, black lines) and amiR-29a LNA (N=6, blue lines). Dashed lines connect pre- and post-MD ODI values of each animal. Solid lines represent the group average. Two-way ANOVA RM, treatment X time, p= 0.0074; post hoc Sidak's test, pre-MD scr vs post-MD scr, ns; pre-MD vs post-MD amiR-29a, p=0.0005.


c ) IOS amplitude ( ± SEM ) in response to contralateral eye ( contra ) and ipsilateral eye ( ipsi ) stimulation . Other conventions as in b ) . amiR - 29a ( N = 6 ) vs scr ( N = 5 ) . Contra eye : two - way ANOVA RM , time X treatment interaction p = 0 . 034 ; post hoc Sidak ' s test ; pre - MD vs post - MD scr , ns ; pre - MD vs post - MD amiR - 29a , p = 0 . 0008 . Ipsi eye : two - way ANOVA RM , time X treatment interaction , ns .

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c) IOS amplitude (± SEM) in response to contralateral eye (contra) and ipsilateral eye (ipsi) stimulation. Other conventions as in b). amiR-29a (N=6) vs scr (N=5). Contra eye: two-way ANOVA RM, time X treatment interaction p=0.034; post hoc Sidak's test; pre-MD vs post-MD scr, ns; pre-MD vs post-MD amiR-29a, p=0.0008. Ipsi eye: two-way ANOVA RM, time X treatment interaction, ns.


d ) Average time course of the IOS response to visual stimulation of the contralateral eye ( contra ) pre - and post - MD ( thick black line ) . Grey area delimited by thin black lines envelops traces from scr animals ( 95 % confidence interval ) ; blue area envelop traces from amiR - 29a animals ( 95 % confidence interval ) . e ) Same as in d ) for ipsilateral eye ( ipsi ) pre - and post - MD responses .

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d) Average time course of the IOS response to visual stimulation of the contralateral eye (contra) pre- and post-MD (thick black line). Grey area delimited by thin black lines envelops traces from scr animals (95% confidence interval); blue area envelop traces from amiR-29a animals (95% confidence interval). e) Same as in d) for ipsilateral eye (ipsi) pre- and post-MD responses.


f ) The effects of MD on Npas IV and Bdnf exIV expression is reported as the ratio between expression levels of deprived and nondeprived cortex for animals treated with scrambled ( scr + MD ) and amiR - 29a ( amiR - 29a + MD ) . Each symbol represent the result of one animal . Error bars represent ± SEM . scr vs amiR - 29a ; t - test ; N = 7 - 9 , Npas4 : p = 0 . 035 ; N = 4 - 5 ; Bdnf exIV : p = 0 . 026 .

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f) The effects of MD on Npas IV and Bdnf exIV expression is reported as the ratio between expression levels of deprived and nondeprived cortex for animals treated with scrambled (scr+MD) and amiR-29a (amiR-29a+MD). Each symbol represent the result of one animal. Error bars represent ±SEM. scr vs amiR-29a; t-test; N=7-9, Npas4: p=0.035; N=4-5; Bdnf exIV: p=0.026.


g ) Scatter plot correlating the changes in protein abundance induced by MD in young mice ( MD CP , x - axis ) with the effects observed in the adult deprived mice treated with amiR - 29a ( MD + LNA _ AD , y - axis ) . Proteins present in both data sets and showing an absolute expression fold change ( FC > 0 . 1 ) are represented . Green symbols correspond to proteins with concordant direction of regulation ; red symbols correspond to discordant regulation . Pearson correlation : r = 0 . 19 , p = 0 . 019 . The number of genes in each quadrant is reported in figure .

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g) Scatter plot correlating the changes in protein abundance induced by MD in young mice (MD CP, x-axis) with the effects observed in the adult deprived mice treated with amiR-29a (MD+LNA_AD, y-axis). Proteins present in both data sets and showing an absolute expression fold change (FC>0.1) are represented. Green symbols correspond to proteins with concordant direction of regulation; red symbols correspond to discordant regulation. Pearson correlation: r=0.19, p=0.019. The number of genes in each quadrant is reported in figure.


a ) Images of WFA - labeled PNNs of scrambled treated visual cortex ( left ) and miR - 29a mimic ( right ) in young mice . Scale bar 60 µm . ( b , c ) Quantification of PNN density ( scr vs mim29a , N = 3 mice per group ; t - test , p = 0 . 20 ) and intensity for each animal ( scr vs mim29a , N = 3 mice per group ; t - test , p = 0 . 014 ) . d ) Cumulative frequency of PNNs staining intensity in scrambled and mimic treated young mice scr ( N = 1438 cells vs mim29a N = 1749 cells , Kolmogorov - Smirnov test , p < 0 . 0001 ) . e ) PNN distribution in classes of intensity in mice treated with scrambled and mimic 29a .

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a) Images of WFA-labeled PNNs of scrambled treated visual cortex (left) and miR-29a mimic (right) in young mice. Scale bar 60 µm. (b,c) Quantification of PNN density (scr vs mim29a, N=3 mice per group; t-test, p=0.20) and intensity for each animal (scr vs mim29a, N=3 mice per group; t-test, p=0.014). d) Cumulative frequency of PNNs staining intensity in scrambled and mimic treated young mice scr (N=1438 cells vs mim29a N=1749 cells, Kolmogorov-Smirnov test, p<0.0001). e) PNN distribution in classes of intensity in mice treated with scrambled and mimic 29a.


f ) Images of WFA - labeled PNNs of scrambled treated visual cortex ( left ) and amiR - 29a LNA ( right ) in adult mice . Scale bar 60 µm . ( g , h ) Quantification of PNN density ( scr vs amiR - 29a , N = 3 mice per group ; t - test , p = 0 . 03 ) and intensity for each animal scr vs amiR - 29a , N = 3 mice per group ; t - test , p = 0 . 047 . i ) Cumulative frequency of PNNs intensity in scrambled and amiR - 29a treated adult mice . scr N = 1883 cells vs amiR - 29a N = 1937 cells , Kolmogorov - Smirnov test , p < 0 . 0001 . j ) PNN distribution in classes of intensity in mice treated with scrambled and amiR - 29a .

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f) Images of WFA-labeled PNNs of scrambled treated visual cortex (left) and amiR-29a LNA (right) in adult mice. Scale bar 60 µm. (g,h) Quantification of PNN density (scr vs amiR-29a, N=3 mice per group; t-test, p=0.03) and intensity for each animal scr vs amiR-29a, N=3 mice per group; t-test, p=0.047. i) Cumulative frequency of PNNs intensity in scrambled and amiR-29a treated adult mice. scr N=1883 cells vs amiR-29a N=1937 cells, Kolmogorov-Smirnov test, p< 0.0001. j) PNN distribution in classes of intensity in mice treated with scrambled and amiR-29a.


k ) Biochemical quantification of C4S in adult amiR - 29a treated mice ( N = 5 ) as compared to scrambled animals ( N = 4 ) . t test , p = 0 . 0008 .

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k) Biochemical quantification of C4S in adult amiR-29a treated mice (N=5) as compared to scrambled animals (N=4). t test, p=0.0008.


l ) Example of Arylsulfatase B ( ArsB ) western blot . Left : The lanes corresponding to the treated ( treat scr or treat amiR - 29a ) and untreated ( untreat scr or untreat amiR - 29a ) cortex ( ctx ) of two amiR - 29a treated and two scr treated mice are reported . Right : quantification of the data shows ArsB upregulation by amiR - 29a . The results are expressed as fold change normalized to untreated cortex . Each symbol represents the result of one animal . N = 4 , scr vs amiR - 29a treated cortex , unpaired t test , p = 0 .

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l) Example of Arylsulfatase B (ArsB) western blot. Left: The lanes corresponding to the treated (treat scr or treat amiR-29a) and untreated (untreat scr or untreat amiR-29a) cortex (ctx) of two amiR-29a treated and two scr treated mice are reported. Right: quantification of the data shows ArsB upregulation by amiR-29a. The results are expressed as fold change normalized to untreated cortex. Each symbol represents the result of one animal. N=4, scr vs amiR-29a treated cortex, unpaired t test, p=0.043


Representative H & E ( top ) and immunofluorescent ( IF ) images ( bottom ) of control and Lats1 / 2 dKO airways at indicated time points post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( for secretory cell , white ) , Acetylated tubulin ( Act - Tub , for ciliated cell , green ) , and DAPI ( blue ) . Scale bar , 100μm . Statistical quantification of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory cells in ( B ) .

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Representative H&E (top) and immunofluorescent (IF) images (bottom) of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (for secretory cell, white), Acetylated tubulin (Act-Tub, for ciliated cell, green), and DAPI (blue). Scale bar, 100μm. Statistical quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells in (B).


Representative IF images of control and Lats1 / 2 dKO airways at indicated time points post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( green ) , AQP5 ( for AT1 cell , white ) , and DAPI ( blue ) . Scale bar , 100μm . Quantification of Scgb1a1 lineage - labeled tdTomato + AQP5 + AT1 cells in ( D ) . Data are presented as mean ± SEM ( n = 5 mice for each group ) .

Representative IF images of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green), AQP5 (for AT1 cell, white), and DAPI (blue). Scale bar, 100μm. Quantification of Scgb1a1 lineage-labeled tdTomato+AQP5+ AT1 cells in (D). Data are presented as mean ± SEM (n=5 mice for each group).


Representative H & E ( top ) and IF images ( bottom ) of control , dKO , and qKO airways at day 21 post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( green ) , AQP5 ( white ) , and DAPI ( blue ) . Quantification of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory cells in ( G ) . Data are presented as mean ± SEM ( n = 3 mice for each genotype ) .

Representative H&E (top) and IF images (bottom) of control, dKO, and qKO airways at day 21 post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green), AQP5 (white), and DAPI (blue). Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells in (G). Data are presented as mean ± SEM (n=3 mice for each genotype).


Representative IF images of control and Lats1 / 2 dKO airways at indicated time points post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CLDN4 ( for DATPs , white ) , CC10 ( G , green ) , AGER ( H , for AT1 cell , green ) , and DAPI ( blue ) . Quantification of Scgb1a1 lineage - labeled tdTomato + CLDN4 + DATPs in ( G and H ) . Data are presented as mean ± SEM ( n = 5 mice for each genotype ) .

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Representative IF images of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CLDN4 (for DATPs, white), CC10 (G, green), AGER (H, for AT1 cell, green), and DAPI (blue). Quantification of Scgb1a1 lineage-labeled tdTomato+CLDN4+ DATPs in (G and H). Data are presented as mean ± SEM (n=5 mice for each genotype).


Representative brightfield and H & E images of 3D organoids . Arrow indicates transitioned 3D organoids retaining DATP and AT1 cells . Scale bars , 1mm for brightfield images , 200μm for H & E images . Representative IF images of secretory , DATP , or AT1 cells in secretory organoids in indicated culture conditions . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( E , green ) , CLDN4 ( E , white ) , AGER ( F , green ) , and DAPI ( blue ) . Quantification of transitioned 3D organoids in 3D normal media ( 3D ) , amino acid - limited Basal MEM media ( BME ) , or affluent repletion of EAA in BME media ( BME + EAA ) with 4 - OHT or EtOH treatment . Representative IF images of YAP and TAZ expression of secretory organoids in indicated culture conditions . Tomato ( for Scgb1a1 lineage , red ) , YAP ( green ) , TAZ ( white ) , and DAPI ( blue ) . Scale bars , 100μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative brightfield and H&E images of 3D organoids. Arrow indicates transitioned 3D organoids retaining DATP and AT1 cells. Scale bars, 1mm for brightfield images, 200μm for H&E images. Representative IF images of secretory, DATP, or AT1 cells in secretory organoids in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), CC10 (E, green), CLDN4 (E, white), AGER (F, green), and DAPI (blue). Quantification of transitioned 3D organoids in 3D normal media (3D), amino acid-limited Basal MEM media (BME), or affluent repletion of EAA in BME media (BME+EAA) with 4-OHT or EtOH treatment. Representative IF images of YAP and TAZ expression of secretory organoids in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), YAP (green), TAZ (white), and DAPI (blue). Scale bars, 100μm.


Representative H & E and IF images of control , Lats1 / 2 dKO , and Raptor ; Lats1 / 2 tKO airways at day 21 post tamoxifen treatment . Tomato ( for Scgb1a1 lineage , red ) , CC10 ( green , upper ) , AGER ( green , lower ) , and DAPI ( blue ) . Scale bar , 100μm .

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Representative H&E and IF images of control, Lats1/2 dKO, and Raptor;Lats1/2 tKO airways at day 21 post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green, upper), AGER (green, lower), and DAPI (blue). Scale bar, 100μm.


Quantification of Scgb1a1 lineage - labeled tdTomato + CC10 + secretory cells ( left ) and tdTomato + AGER + AT1 cells ( right ) in ( E ) . Data are presented as mean ± SEM ( n = 2 - 3 mice for each genotype ) . * * * p < 0 . 001 ( Student ' s t - test ) .

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Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells (left) and tdTomato+AGER+ AT1 cells (right) in (E). Data are presented as mean ± SEM (n=2-3 mice for each genotype). ***p<0.001 (Student's t-test).


Measurement of L - amino acid or essential amino acids ( leucine , isoleucine , valine ) contents in Scgb1a1 lineage - labeled tdTomato + organoids derived from control , Lats1 / 2 dKO or Raptor ; Lats1 / 2 tKO mice . Data are presented as mean ± SEM ( n = 3 technical replicates for each genotype ) . * p < 0 . 05 , * * p < 0 . 001 ( Student ' s t - test ) .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Measurement of L-amino acid or essential amino acids (leucine, isoleucine, valine) contents in Scgb1a1 lineage-labeled tdTomato+ organoids derived from control, Lats1/2 dKO or Raptor;Lats1/2 tKO mice. Data are presented as mean ± SEM (n=3 technical replicates for each genotype). *p<0.05, **p<0.001 (Student's t-test).


Western blot images for the expression of YAP , ATF4 , and LAT1 / Slc7a5 in control or YAP5SA overexpressed C22 airway epithelial cell line . Knockdown of ATF4 was induced by using lentiviral - mediated inducible scramble shRNA ( shScramble , control ) or shATF4 .

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Western blot images for the expression of YAP, ATF4, and LAT1/Slc7a5 in control or YAP5SA overexpressed C22 airway epithelial cell line. Knockdown of ATF4 was induced by using lentiviral-mediated inducible scramble shRNA (shScramble, control) or shATF4.


Concentration of L - amino acids ( L - AA , left ) or EAA ( right ; leucine , isoleucine , valine ) in cell lines in ( A ) . Data are presented as mean ± SEM ( n = 3 biological replicates for each group ) . * p < 0 . 05 , NS = not significant ( Student ' s t - test ) .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Concentration of L-amino acids (L-AA, left) or EAA (right; leucine, isoleucine, valine) in cell lines in (A). Data are presented as mean ± SEM (n=3 biological replicates for each group). *p<0.05, NS=not significant (Student's t-test).