METHOD FOR DIFFERENTIATING PLURIPOTENT STEM CELLS INTO UNDERLYING CONNECTIVE TISSUE FIBROBLASTS OF AN EPITHELIUM

Abstract

The invention relates to a method for differentiating human pluripotent stem cells into fibroblasts, characterized in that the human pluripotent stem cells are cultured on an adherent system in the presence of a medium that is suitable for culturing fibroblasts and in the absence of feeder cells.

Claims

1. A method for differentiating human pluripotent stem cells into fibroblasts, comprising the step of culturing the human pluripotent stem cells on an adherent system in the presence of a medium that is suitable for culturing fibroblasts and in the absence of feeder cells.

2. The method according to claim 1, wherein the fibroblasts are dermal fibroblasts.

3. The method according to claim 1, wherein the fibroblasts are at least 90% papillary fibroblasts.

4. The method according to claim 1, wherein the medium that is suitable for culturing fibroblasts comprises insulin, hydrocortisone, epidermal growth factor (EGF) and fibroblast growth factor (FGF).

5. The method according to claim 1, wherein the medium that is suitable for culturing fibroblasts is supplemented with Bone Morphogenetic Protein 4 (BMP-4).

6. A method for differentiating human pluripotent stem cells into fibroblasts comprising the steps of: (a) optionally, forming and culturing aggregates or clusters of said human pluripotent stem cells on an adherent system to support cell attachment and growth in the presence of a medium that is suitable for culturing human pluripotent stem cells; (b) culturing the human pluripotent stem cells or adherent aggregates or clusters of said human pluripotent stem cells on a cell culture surface coated with a defined protein matrix coating in the presence of a medium that is suitable for culturing fibroblasts; (c) differentiating the human pluripotent stem cells into fibroblasts by culturing on a protein matrix in the presence of a medium that is suitable for culturing fibroblasts, for 12 to 16 days; d) optionally, maturing the fibroblasts obtained in step c) at least during passaging on a protein matrix in the presence of a medium that is suitable for maturation; e) optionally, maturing the fibroblasts obtained in step d) during passaging in the presence of a medium that is suitable for maturation, with or without the protein matrix; and f) selecting cells obtained at step (c), (d) or (e) to obtain a homogeneous population of fibroblasts.

7. The method according to claim 6, in which step b) of amplifying the human pluripotent stem cells comprises manually passaging the human pluripotent stem cells.

8. The method to claim 6, in which step b) of amplifying the human pluripotent stem cells comprises enzymatically passaging the human pluripotent stem cells.

9. The method according to claim 6, wherein the medium that is suitable for culturing fibroblasts is supplemented with Bone Morphogenetic Protein 4 (BMP-4) at a concentration of between 0.1 and 0.5 nM.

10. A method for preparing a connective tissue, comprising culturing fibroblasts obtained according to the method of claim 1 under conditions suitable for the formation of connective tissue.

11. A method for the evaluation of compounds comprising the step of contacting the fibroblasts obtained according to the method of claim 1 under cell culture conditions suitable for the evaluation of the compounds.

12. A method for producing proteins for therapeutic use comprising the step of culturing the fibroblasts obtained according to the method of claim 1 under cell culture conditions suitable for production of the proteins.

13. The method of claim 6 wherein step c) is performed for 13 to 15 days.

14. The method of claim 6 wherein step c) is performed for 14 days.

15. The method according to claim 6, in which Bone Morphogenetic Protein-4 (BMP-4) is added at a concentration of between 0.2 and 0.4 nM on D1 and D4.

16. The method according to claim 6, in which Bone Morphogenetic Protein-4 (BMP-4) is added at a concentration of about 0.27 nM on D1 and D4.

17. The method of claim 10, wherein the connective tissue is dermis.

18. The method of claim 10, wherein the connective tissue is 3D skin.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0071] Other features, details and advantages of the invention will become apparent on examining the appended Figures.

[0072] FIG. 1

[0073] FIG. 1 represents protocols for the differentiation of hESC cells into fibroblasts.

[0074] FIG. 2

[0075] FIG. 2 represents the protocol for differentiating hESC cells into fibroblasts according to the invention.

[0076] FIG. 3

[0077] FIG. 3 represents the subtypes of fibroblasts in the skin and the associated markers.

[0078] FIG. 4

[0079] FIG. 4 represents the quality control of hPSC-derived fibroblasts.

[0080] FIG. 5

[0081] FIG. 5 represents the comparison of FRC9 and of primary fibroblasts.

[0082] FIG. 6

[0083] FIG. 6 represents the differentiation into fibroblasts starting with F-1432 IPS.

[0084] FIG. 7

[0085] Western blotting of total proteins following the differentiation into myofibroblasts of fibroblasts obtained from iPSCs, adult fibroblasts, neonatal fibroblasts and fetal fibroblasts.

[0086] FIG. 8

[0087] Immunofluorescence analysis of α-SMA-positive cells of cells differentiated into myofibroblasts.

EXAMPLES

Example 1: Materials and Methods

[0088] Pluripotent cell line used: hES line (RC9, Roslin Cells)

[0089] Primary fibroblast culturing:

[0090] The various types of primary fibroblasts used are: [0091] Human Dermal Fibroblasts pooled foreskin: F-Adultes (HDFp, CELLnTEC) [0092] Normal Human Dermal Fibroblasts Fetal Foreskin: HDFF (Cell Application, 106-05f) [0093] Normal Human Dermal Fibroblasts neonatal Foreskin: HDFN (Promocell, C-12300) [0094] Normal Human Dermal Fibroblasts adult: HDFA (Promocell, C-12302)

[0095] Clinical-grade differentiation protocol according to the invention:

[0096] Colonies of hPSC cells are cut with a needle and 1 clump/cm.sup.2 is seeded in containers coated with L7 matrix in Stem Pro medium supplemented with stabilized FGF-2 for 24 hours (DO). The next day, the culture medium is changed to CnT-Prime-Fibroblast medium (F-CnT) supplemented with defined and secured fetal calf serum (final concentration: 5%) up to D14. BMP-4 treatment at 0.27 nM is applied on D1 and D4.

[0097] From D14/p0, when confluence is reached, the cells are passaged by trypsin treatment for 5 min at +37° C. The cells undergoing differentiation are reseeded at 50 000 cells per cm.sup.2 up to D35/p2 on L7 matrix. The cells are thus ready to be used (banking, clinical, other uses).

Example 2: Differentiation Protocol

[0098] Several differentiation conditions were tested and compared.

[0099] Condition 1 (FD1 or DF1) is performed with the same media as the protocol of Shamis et al. up to p3/D28, but without feeder and on the L7 matrix.

[0100] The media NHK and SCES are described in the publication by Hewitt K., Shamis Y., Carlson M., Aberdam E., Aberdam D., Garlick J. Three-dimensional epithelial tissues generated from human embryonic stem cells. Tissue Eng. Part A. 2009 15(11): 3417-3426.

[0101] The NHK medium comprises a mixture of DMEM:F12 3:1, 5% FCII, 0.18 mM adenine, 8 mM HEPES, 0.5 μg/mL hydrocortisone, 10.sup.−10 M cholera toxin, 10 ng/mL EGF and 5 μg/mL insulin.

[0102] The SCES medium comprises a mixture of DMEM:F12 1:1, 5% FCII and 1% nonessential amino acids.

[0103] Condition 2 (DF2 or FD2) is performed with the same media as the original protocol (DF1), i.e. the Shamis protocol but without feeder and on the L7 matrix, up to p1/D14 and then the F-CnT 5% medium up to p3/D28.

[0104] FD3 is performed with the same media as the original protocol up to p0/D7 and then 5% F-CnT medium up to p3/D28.

[0105] FD4: is performed solely with 5% F-CnT medium up to p3/D28.

[0106] On D21/p2, the cells of all the conditions are frozen and stored in nitrogen until the time of use.

[0107] For quality control, the cells are thawed and then amplified in F-CnT medium (without the addition of 4% serum as for differentiation) and amplified to 100% confluence. The morphology of the cells obtained is similar irrespective of the differentiation conditions FD1, FD2, FD3 and FD4 but are different than the primary cells (F-Adult). After passaging, the cells are analyzed by FACS so as to identify the populations obtained.

[0108] Several markers are analyzed: fibroblast characterization markers: CD29 (integrin p1), CD73/CD166 (mesenchymal stem cell markers) and “Fibro” (fibroblast markers).

[0109] Analysis of the expression of all these markers on primary adult cells (F-Adults) shows that the “fibroblast” profile is reflected by an expression of: CD29/CD166/FIBRO of greater than 90% and CD73 of greater than 75%.

[0110] Analysis of the four differentiation conditions FD1, FD2, FD3 and FD4 shows that only one differentiation reaches the specifications obtained with adult fibroblasts: the FD4 condition with an expression of CD29 at 99.5%, CD73 at 89%, CD166 at 95.4% and FIBRO at 97.5%.

[0111] This “FD4” condition is thus validated for the differentiation protocol. It is also the simplest since there is only one medium reference for the whole process, supplemented with 5% serum for the “differentiation” step between D1 and freezing at p2.

[0112] For the subsequent tests, the density was increased to 50 000 cells/cm.sup.2 at the end of differentiation p0 and p1 and then from p2 onwards to between 5000-10 000 cells/cm.sup.2. At 50 000 cells/cm.sup.2 there are more adherent cells than at 5000-10 000 cells/cm.sup.2 but the level of non-adherent cells is also higher. This differentiation end step (p0) thus appears to be a selection step for fibroblast “progenitors”. The sorting is done on the basis of adhesion: non-adhered cells are not sufficiently mature or are not fibroblast progenitors. This enables the future fibroblasts to be purified and the population to be made homogeneous in the course of the following passages.

[0113] These differentiation and passaging end densities made it possible to obtain hESC-derived fibroblasts proliferating to at least p7 (subsequent passages not tested) with no significant decrease in yield at each passage.

[0114] The established differentiation protocol thus consists of several steps:

[0115] On D0, colonies of hPSC cells are cut into clumps with a needle under a binocular loupe. Approximately 1 clump/cm.sup.2 is seeded in stoppered containers (flasks) coated with L7 coating and in StemPro hESC medium with 10 ng/mL of stabilized FGF-2 (hPSC culture conditions) to allow hESC adhesion.

[0116] From D1 to D7, the StemPro/FGF2 medium is replaced with ready-to-use 1% F-CnT medium which is supplemented with an additional 4% FCS (to give a final concentration of 5%). When the same experiments were performed with 1% FCS, significant cell death was observed and the cells were less well differentiated into fibroblasts.

[0117] From D1 to D7, the 5% F-CnT medium is supplemented with BMP-4 (0.273 nM) to inhibit the neural pathway. Two pulses of BMP-4 are performed on D1 and D4. BMP-4 is present from D1 to D7 inclusive.

[0118] From D8 to D13, the cells are maintained with 5% F-CnT medium to allow proliferation and differentiation of the hPSCs.

[0119] On D14 (p0), the cells rinsed with PBS1× are placed in contact with 0.05% EDTA trypsin for 5-10 min. The trypsin action is stopped with 10% FCS and the cells are filtered through a 40 μm sieve to remove the undissociated cell clusters. The cells are then seeded at 50 000 cells/cm.sup.2 in stoppered containers (flasks) coated with L7 coating and in 5% F-CnT medium.

[0120] The morphology of the FRC9 obtained after the RC-9 hESC differentiation process is very similar to that of the adult fibroblasts (F-Adult) and no longer resembles that of the starting RC-9 hESCs. Analysis of the expression of the FIBRO (or Fibroblast) marker shows an absence of expression of this marker on the RC-9 hESCs and an expression of 96.6% on the FRC9s, close to the level of the adult fibroblasts at 99.9%. In addition to this marker, the cells also express vimentin (fibroblast marker) and collagen I (secreted by mature fibroblasts) in the cytoplasm. Collagen I does not appear to be secreted by the FRC9 or F-Adults as there is no deposit. ELISA tests could be performed to observe the secretion of procollagen I.

Example 3: Phenotypic Characterization of hPSC-Derived Reticular and Papillary Fibroblasts

[0121] The literature shows that different types of fibroblasts exist and that, depending on their location within the same tissue or in a different tissue, they express specific markers (FIG. 3):

[0122] Fibronectin: involved in organizing the extracellular matrix (ECM)

[0123] Serpin H1 (HSP47): involved in collagen synthesis in dermal fibroblasts Decorin (DEC): involved in the assembly of collagen fibrils

[0124] Collagen I and III (COL1A and COL3A): ECM proteins

[0125] Podoplanin (PDPN): involved in ECM remodeling by papillary fibroblasts

[0126] Calponin 2 (CNN2): involved in inhibiting reticular fibroblast proliferation

[0127] Versican (VCAN): ECM protein secreted by reticular fibroblasts

[0128] A wide range of markers from the literature was tested by qPCR, ICC and FACS to determine the exact identity of the fibroblast population produced by the differentiation protocol of the present invention (FIG. 4).

[0129] Gene expression analysis by qPCR (normalized on the 18S gene and RC-9 cells) shows that the RC9-derived fibroblasts express the markers HSP47, DEC, COL1A, COLA3, PDPN and NTN1 at high levels similar to those of adult fibroblasts. The reticular markers VCAN and CNN2 are not or are only sparingly expressed by FRC9p2 and adult fibroblasts. A slight increase in the expression of the reticular markers VCAN and CNN2 on FRC9p7 is observed.

[0130] FACS analysis shows that FRC9 and F-Adults express the CD73/CD166 markers at more than 80% and the “Fibroblast” marking at more than 96%.

[0131] Analysis by ICC shows that FRC9s and F-Adults have similar profiles:

[0132] HSP47 is indeed expressed in the endoplasmic reticulum,

[0133] Collagens I and III are also expressed in the cytoplasm with a location near the endoplasmic reticulum,

[0134] Fibronectin is expressed in the cytoplasm and there are deposits on the outside of the cells, which shows that the protein has been secreted,

[0135] Podoplanin is expressed throughout the cytoplasm,

[0136] Calponin 2 is expressed by very few cells.

[0137] The results show that the fibroblast differentiation protocol makes it possible to obtain dermal fibroblasts due to the expression of serpinH1/HSP47 and that the population obtained is mainly papillary due to the strong expression of podoplanin and the weak expression of calponin 2 which is only observed on a few cells. The F-Adults and FRC9s also express collagens I/III, but these proteins do not appear to be secreted since no deposits are detected, unlike fibronectin which is expressed and secreted. Collagen secretion by F-Adults and FRC9s appears to require different culture conditions.

Example 4: Comparison of hPSC-Derived Fibroblasts and Primary Fibroblasts

[0138] Still for the purpose of characterizing the cells and improving their quality control, a comparative study between FRC9 and three primary fibroblast lines (fetal, neonatal and adult) was performed (FIG. 5).

[0139] qPCR analysis shows that the expression profile of the DECORIN, COL1A, COL3A, PDPN, NTN1 and VCAN genes is similar between the three primary fibroblast types and FRC9. SERPIN H1 gene expression is similar to that of the adult cells but lower than that of the neonatal and fetal fibroblasts. The CNN2 gene is weakly expressed by the FRC9s and adult fibroblasts and is not expressed in the neonatal and fetal fibroblasts.

[0140] FACS analysis shows that the adult, neonatal and FRC9 fibroblasts have a similar profile for the markers “fibroblast”, vimentin and podoplanin with an expression of more than 97%. The fetal fibroblasts have a lower expression for vimentin marking with 90.9%. For the PDGFR marking (marking of reticular cells, involved in cell differentiation), the profile is globally observed to be weakly expressed, with an expression of less than 13% for all the lines.

[0141] Analysis by ICC shows that the three types of primary fibroblasts and FRC9 strongly and homogeneously express the markers Serpin H1, Collagen I, podoplanin with localization to the endoplasmic reticulum for the Serpin H1 and Collagen I markers. The TG2 marker (Transglutaminase 2, involved in the structure of the ECM), is very sparingly expressed by the four types of fibroblasts.

[0142] The analyses performed with the three types of fibroblasts do not make it possible to differentiate them and therefore to apply an “adult”, “neonatal” or “fetal” profile to the FRC9s.

Example 5: Differentiation of iPSC Line PC1432 into Fibroblasts

[0143] The process used for the RC 9 line also works for iPS.

[0144] FACS analysis of iPS shows an expression profile similar to that of HDNF with more than 99% expression of the markers “Fibroblast”, podoplanin and vimentin.

[0145] The differentiation process thus makes it possible to obtain fibroblasts from iPS.

Example 6: Characterization of Fibroblasts Derived from iPSC1432 Cells

[0146] The objectives of this study were, firstly, to evaluate the capacity of fibroblasts derived from iPSCs 1432 cells to differentiate into myofibroblasts after induction with TGF-beta and, secondly, to provide additional information regarding their characterization, notably on the developmental stage of these fibroblasts.

[0147] Indeed, obtaining early-stage fibroblasts would be advantageous because fetal and neonatal fibroblasts have faster and more efficient healing capacity than adult fibroblasts, notably for fetal fibroblasts for which skin repair in the early stages of gestation is rapid and without scarring.

[0148] To date, there are no specific markers for each stage (fetal, neonatal and adult) that allow them to be differentiated in vitro, but a comparative study of their respective functions can shed light on this matter. Publications have notably shown that fetal fibroblasts secrete much less TGF-beta 1 (which results in a weaker inflammatory response) than adult fibroblasts, but on the other hand that they do secrete much more collagen, notably type III and IV collagens (Larson, Longaker et al. 2010; Kishi, Okabe et al. 2011; Tang, Chen et al. 2014). The major difference between fetal and adult skin is the composition of the dermis (Coolen, Schouten et al. 2010). Where neonatal skin is histologically indistinguishable from adult skin, fetal skin shows an absence of elastin up to 22 weeks of gestation, but a larger amount of fibronectin. The fetal fibroblast has an intrinsic ability to synthesize a dermal extracellular matrix that is superior to that of adult fibroblasts, which makes it possible to generate a better-organized dermis at the sites of injury (more secreted matrix, but also more secreted metalloproteases for better remodeling).

[0149] In other studies, it was shown that proliferation did not differ between the neonatal and the adult fibroblasts, but their migratory potential was different (Mateu, Živicová et al. 2016). Furthermore, the authors show a larger pool of α-SMA (+) and Nestin (+) cells in the neonatal population compared to adults.

[0150] Differentiation into Myofibroblasts, Western Blotting (WB) Analysis: Comparison with Adult, Neonatal and Fetal Fibroblasts.

[0151] In this experiment, differentiation into myofibroblasts (induced by adding TGF-beta 1 to the culture medium) was performed in two different culture media: CnT-PR-F medium and DMEM 10% FCS medium, which is the medium conventionally used in the literature for performing this type of experiment. At the end of the differentiation, the total proteins of each condition were collected and analyzed by WB. The α-SMA protein is the best known marker of myofibroblasts. It is overexpressed when fibroblasts differentiate. Another feature of these myofibroblasts is an increase in cell contractility through myosin activation characterized by serine 19 phosphorylation. This specific form is also analyzed in this WB (the term P-MLC meaning phospho Myosin Light Chain).

[0152] Primary fibroblasts (adult and neonatal) differentiate into myofibroblasts only under the DMEM 10% FCS condition. The CnT-PR-F medium does not allow differentiation of these primary fibroblasts. Fibroblasts derived from iPSCs 1432 (FIBs IPSCs or FIBs 1432) and fetal fibroblasts are able to differentiate in both media, but with stronger differentiation in DMEM medium (weak increase in SMA expression in CnT-PR-F+TGF-beta medium). On the other hand, the DMEM 10% FCS medium appears to induce a basic differentiation of iPSCs and primary FIBs compared with CnT-PR-F medium independently of TGF-beta.

[0153] The increase in phospho-MLC2 after TGF-beta treatment is clearly visible by WB. Compared with α-SMA, it is visible for all the lines irrespective of the culture medium used (small increase for adults in CnT-PR-F but visible after quantification).

[0154] Overall, for the marker of contractility (P-MLC2) and that of the presence of myofibroblasts (SMA), it appears that the iPSC FIBs have a profile that is different from that of the adult and neonatal fibroblasts but similar to that of the fetal fibroblasts (cf. FIG. 7).

[0155] Differentiation into myofibroblasts, analysis by Immunofluorescence:

[0156] In parallel to the WB, the same differentiation experiment was performed for immunofluorescence analysis. The following images show the α-SMA-positive cells. The results follow those observed by WB, i.e. there are no α-SMA (+) cells in the CnT-PR-F+TGF-beta medium for adults and neonatals in contrast with iPSCs FIBs and fetal fibroblasts. In contrast, numerous SMA (+) cells are observed in DMEM+TGF-beta for the four lines. The adult and neonatal fibroblasts appear to be able to differentiate only in DMEM 10% FCS medium, whereas the 1432 FIBs and fetal fibroblasts are able to differentiate in both media as observed in WB.

[0157] On the basis of these experiments, it appears that 1432 FIBs have a differentiation profile into myofibroblasts that is similar to that of fetal fibroblasts.