Hepatic Stem-Like Cells for the Treatment and/or the Prevention of Liver Disorders

Abstract

The present invention relates to a population of cells comprising hepatic stem-like cells and therapeutic use thereof, for the treatment and the prevention of fulminant liver disorders. The hepatic stem-like cells according to the invention may be safely and reproducibly generated from pluripotent stem cells. In addition, although the hepatic stem-like cells according to the invention do not display the phenotype of physiologically mature hepatic cells, as they are lacking the albumin expression marker (ALB), they may still be transplanted in a diseased liver with acute failure, rescue the diseased liver and promote liver regeneration. Moreover, various protocols of preparation of hepatic stem-like cells according to the invention may be implemented, all resulting in high quality and high yield of production. Finally, the hepatic stem-like cells according to the invention may be cryopreserved and may also be prepared as spheroid particles.

Claims

1. An isolated population of cells, comprising at least 5% of hepatic stem-like cells expressing the alpha-ftoprotein marker (AFP+) and not expressing the albumin marker (ALB), or an extract thereof.

2. The isolated population of cells according to claim 1, wherein the hepatic stem-like cells are further expressing the T-Box Transcription Factor 3 marker (TBX3+) and/or the Hepatocyte Nuclear Factor 4 Alpha marker (HNF4A+), preferably the T-Box Transcription Factor 3 marker (TBX3+) and the Hepatocyte Nuclear Factor 4 Alpha marker (HNF4A+).

3. The isolated population of cells according to claim 1, wherein the hepatic stem-like cells are cryopreserved.

4. A particle, in particular a spheroid, comprising the isolated population of cells, or an extract thereof, according to claim 1.

5. A suspension comprising the isolated population of cells, or an extract thereof, according to claim 1.

6. A pharmaceutical composition comprising (i) the isolated population of cells, or an extract thereof, according to claim 1, and (ii) a pharmaceutically acceptable vehicle.

7. A medical device comprising the isolated population of cells, or an extract thereof according to claim 1.

8. A non-human animal model comprising the population of cells, or an extract thereof, according to claim 1, wherein the population of cells are heterologous.

9. (canceled)

10. A method of preventing and/or treating a fulminant liver disorder in a subject in need thereof comprising administering the population of cells, or an extract thereof, according to claim 1.

11. The method according to claim 10, wherein the fulminant liver disorder is an acute liver failure (ALF) or an acute chronic liver failure (ACLF).

12. The method according to claim 11, wherein the ACLF is associated with a liver disease selected in the group consisting of the non-alcoholic steatohepatitis (NASH); alcoholic hepatitis; viral-induced hepatitis; a cryptogenic liver disease; a malignant liver disease, such as hepatocellular carcinoma and cholangiocarcinoma; autoimmune hepatitis, a vascular liver disease, such as Budd-Chiari syndrome; a cholestatic liver disease; and an inherited metabolic liver disease, such as, Wilson's disease and an urea cycle disorder.

13. The population of cells, or an extract thereof, according to claim 1, wherein the population of cells is cryopreserved.

14. An in vitro method for screening a drug, said method comprising the steps of: a. providing population of cells according to claim 1; b. contacting said population of cells or extract thereof, from step (a), with a drug candidate; c. measuring one or more biological parameter(s) and optionally comparing said one or more biological parameter(s) with one or more reference parameter(s); d. determining whether the drug candidate is of therapeutic and/or diagnostic interest.

15. A kit for treating and/or preventing a fulminant liver disorder, said kit comprising: a. a population of cells, or an extract thereof according to claim 1; and b. a mean to administer said cells or extract thereof, population or extract thereof, or particle, or suspension or pharmaceutical composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0347] FIGS. 1A-D are histograms showing the relative levels of mRNAs of markers, determined by RT-qPCR, encoding pluripotency (OCT4) (Panel A), definitive endoderm (SOX17) (Panel B)), and hepatic progenitor genes (HNF4A) (Panel C), AFP (Panel D) at day 11 (pStemHeps). The relative gene expression was calculated using the 2-Ct quantification method after normalization to GAPDH values and expressed as fold of levels found in undifferentiated hESCs cells (D0). Relative gene expression levels are in Log 10 scale for SOX17 (Panel B), HNF4A (Panel C) and AFP (Panel D). * P<0.05.

[0348] FIGS. 2A-D are photographs and plots showing the expression or the non-expression of different key markers through hepatic differentiation of hESCs from day 0 (D0), day 5 (D5), day 11 (D11) by immunofluorescence assays (OCT4, FOXA2, AFP and ALB) (Panel A), by flow cytometry for SOX17/HNF4A (Panel B) and for CXCR4 (Panel C) and by ELISA measuring AFP secretion in cell supernatant (Panel D). HPH: human primary hepatocytes, * P<0.05.

[0349] FIGS. 3A-C are photographs and graph showing the expression of ALB, AFP and HNF4A by immunofluorescence test (Panel A) at D21 after hepatic differentiation of hESC into HLCs, cell morphology of pStemHeps and HLCs examined by phase contrast microscopy (Panel B) and secretion of ALB in cell supernatant by HLCs measured by ELISA test (Panel C). * and ** P<0.05.

[0350] FIG. 4 is a plot showing the percentage survival of mice that did not received (plot 1, control animals with no APAP, n=10) or received (plots 2 and 3) 400 mg/kg body weight of acetaminophen (APAP) and further received (plot 2, APAP+pStemHeps, n=10) or not (plot 3, control ALF animals with APAP only, n=10) transplantation of 110.sup.6 frozen pStemHeps that were prepared accordingly to the protocol A, as described in Example 1.

[0351] FIG. 5 is a plot showing the alanine aminotransferase (ALAT) levels (expressed in U/L) in the blood of mice that did not received APAP (NO APAP only, n=5), received only APAP (APAP only, n=6) or received APAP and 110.sup.6 frozen pStemHeps (APAP+pStemHeps, n=8), treated as in FIG. 4. Serum ALAT was measured at 24 hours post-cell injection. * and ** P<0.05.

[0352] FIG. 6 is a plot showing the percentage of healthy tissue in the three groups of mice treated as in FIG. 4 (APAP+pStemHeps, n=10; APAP only, n=8; No APAP, n=5) and at 24 hours post-cell injection. Each dot corresponds to an animal. * and ** P<0.05.

[0353] FIG. 7 is a plot showing the levels of production of human AFP in animal serum measured by ELISA in animals receiving only APAP (APAP only, n=3) and receiving APAP and pStemHeps (APAP+pStemHeps, n=9), treated as in FIG. 4. Human AFP levels were determined at 24 h post-transplantation of 110.sup.6 frozen pStemHeps. Each dot corresponds to an animal. * P<0.05.

[0354] FIG. 8 is a plot showing the presence of human Alu sequences in liver biopsies collected from the three groups of mice treated as in FIG. 4 and at 24 h after transplantation of 110.sup.6 frozen pStemHeps as measured by PCR analysis and PCR amplicons detection by capillary electrophoresis. The arrow corresponds to the Alu PCR amplicon. Lanes 1-4: APAP+pStemHeps; lanes 5-6: APAP only; lanes 7-8: No APAP, no cells; lane 9: CTL+(liver of a mice transplanted with human cells); lane 10: Blank; lane 11: ladder.

[0355] FIG. 9 is a histogram showing the relative levels of mRNAs of characteristic cell markers, determined by RT-qPCR in pStemHeps that were prepared accordingly to the protocol A, B or C as described in Example 2. The relative gene expression levels were expressed as fold of those found in undifferentiated hESCs. They are significantly different from hESCs. Relative gene expression levels are in Log 10 scale.

[0356] FIG. 10 is a histogram showing the expression or non-expression of mRNAs for some characteristic cell markers, determined by DGE-Seq by pStemHeps that were prepared accordingly to the protocol A (n=11; dark grey bars) or protocol B (n=3; light grey bars) as described in Example 2. Depicted expressed genes are defined as genes for which the number of mRNA are above 200 molecules per million of total mRNA molecules (highly expression, set 1), and genes for which the number of mRNA is between 5 and 200 molecules per million of total mRNA molecules and the fold induction change versus undifferentiated hESCs (n=14) is above 5 (lower expressed genes as compared to set 1). Depicted non expressed gene are defined as genes for which the number of mRNA is zero or below 5 molecules per million of total mRNA molecules.

[0357] FIGS. 11A-B are a plot and photographs showing the expression of AFP and non-expression of ALB by pStemHeps that were prepared accordingly to the protocol A, B or C by ELISA test (Panel A) and the expression of AFP, CK19, EPCAM, FOXA2, HNF4A, K167, SOX17 and non-expression of ALB by immunofluorescence test (Panel B), by pStemHeps that were prepared accordingly to the protocol C, as described in Example 2.

[0358] FIG. 12 is a plot showing the percentage survival of mice (n=10 in each group) as in FIG. 4, except that frozen pStemHeps were prepared accordingly to protocol B, as described in Table 2 of Example 2. Plot 1: CTRL NO APAP, Plots 2: APAP+pStemHeps, plot 3: APAP only.

[0359] FIGS. 13A-B are plots showing the percentage survival of mice as in FIG. 12, except that mice have undergone surgery to remove of the liver just before transplantation of pStemHeps that were prepared accordingly to protocol B (Panel A) of protocol C (Panel B), as described in Table 2 of Example 2. Panel A: plot 1: APAP+pStemHeps (n=5); plot 2: APAP only (n=10); Panel B: plot 1: APAP+pStemHeps (n=5); plot 2: APAP only (n=5).

[0360] FIG. 14A-C is a set of plot and photographs showing (Panel A) the percentage survival of mice as in FIG. 4, except that frozen pStemHeps were prepared accordingly to protocol C, as described in Table 2 of Example 2. Plot 1: APAP+pStemHeps (n=4), plot 2: APAP only (n=5); (Panels B-C) the number of proliferating cells in liver sections by immunohistochemistry using antibodies against K167 (MKI67) at 24 hours after injection of 110.sup.6 pStemHeps in animals that did received 700 mg/kg body weight of acetaminophen (APAP+ Cells; Panel B) or in untreated animals that did received APAP only (control APAP; Panel B). A K167 (MKI67) positive cell nuclei appears in dark dot. Original magnification 10.

[0361] FIGS. 15A-B are plots showing the percentage survival of mice received 1500 mg/kg body weight of thioacetamide (TAA) and further received (plot 1, TAA+pStemHeps, n=9 for Panel A and n=5 for Panel B) or not (plot 2; TAA only, n=10) transplantation of 110.sup.6 frozen pStemHeps that were prepared accordingly to protocol C, as described in Table 2 of Example 2. Treated mice further received (Panel A) or not (Panel B) 1 mg/kg of tacrolimus (daily for 5 days and beginning 24 hours before cell injection).

[0362] FIG. 16 is a plot showing the levels of production of human AFP measured by ELISA in TAA-intoxicated C57BL/6 mice that did not received (CTRL), or that received 110.sup.6 frozen pStemHeps that were prepared accordingly to protocol C, as described in Table 2 of Example 2. Human AFP levels were determined in mice after 24 h post-transplantation. Each dot corresponds to an animal. * P<0.05.

[0363] FIGS. 17A-C are photographs showing the generation of spheroids prepared from freshly-prepared (Panel A) and from cryopreserved pStemHeps (Panel B) after plating and 2 days of culture into Aggrewell plates, as described in example 3. Panel C is a photograph showing viability of spheroids, as measured by Live/Dead assay immunofluorescent test. LIVE: esterase activity, DEAD: staining of Dead cells with ethidium homodimer-1, NUCLEI: cell nucleus staining with Hoechst 33342, LIGHT: phase contrast microscopy.

[0364] FIG. 18 is a histogram showing the relative levels of mRNAs of markers in freshly-prepared (pStemHep FRESH) or cryopreserved (pStemHep FROZEN) pStemHeps cultured as indicated in example 2 and in spheroids prepared from freshly-prepared (SPHE FRESH) or from cryopreserved pStemHeps (SPHE FROZEN) generated and cultured for 2 days as indicated in example 3, as determined by RT-qPCR, SOX17, HNF4A, and AFP. Relative gene expression levels are in Log 10 scale. All relative gene expression levels were significantly different from those of undifferentiated hESCs.

[0365] FIG. 19 is a photograph showing the expression of AFP, CK19, FOXA2, HNF4A and SOX17 and non-expression of ALB by 2-days cultured spheroids prepared from pStemHeps measured by immunofluorescent test.

[0366] FIGS. 20A-B are plots showing the expression of AFP and non-expression of ALB by spheroids (SPHE) that were prepared from pStemHeps and cultured for 2 days in Aggrewell 400 by ELISA tests. pStemHeps and HLCs (generated as indicated in example 1) were used as positive controls for secretion of human AFP (Panel A) and human ALB (Panel B), respectively. * P<0.05.

[0367] FIG. 21 is a plot showing the percentage survival of mice (n=5) in each group) as in FIG. 4, except that frozen pStemHeps were prepared accordingly to protocol C, as described in Table 2 of Example 2, and in C57BL6 mice that did received 700 mg/kg body weight of acetaminophen and further transplanted with 210.sup.7 pStemHeps in the peritoneal cavity (plot1) or not (plot 2).

[0368] FIG. 22 is a plot showing the levels of in vivo production of human AFP measured by ELISA in 3 groups of C57BL/6 mice that did not received (CTRL, n=3), or that received cell-free/pre-molded alginate hydrogel transplanted in the peritoneal cavity (Alginate, n=5), or pre-molded alginate hydrogel containing 2 days-cultured spheroids that were prepared from 110.sup.7 pStemHeps and transplanted in the peritoneal cavity (SPHE Alginate, n=3). Human AFP levels were determined in mice after 24 h post-transplantation. Each dot corresponds to an animal. * P<0.05.

[0369] FIG. 23 is a photograph showing high viability of spheroids that were transplanted and harvested from animals depicted in FIG. 21 at day 8 post-transplantation, as measured by Live/Dead assay immunofluorescent test. After 8 days in the mice peritoneal cavity, most of cells in SPHE have high esterase activity (LIVE) and only few cells were positive to ethidium homodimer-1 (DEAD). Cell nucleus were stained with Hoechst 33342 (NUCLEI).

[0370] FIGS. 24A-B are histograms showing the relative levels of mRNAs of key markers of hepatic differentiation, i.e., AFP (Panel A) and HNF4A (Panel B), in spheroids that were prepared from pStemHeps cultured in vitro for 8 days (SPHE D8 in vitro) and in spheroids embedded in alginate hydrogel that were transplanted and harvested from animals depicted in FIG. 21 at day 8 post-transplantation (SPHE D8 Alginate), as determined by real-time RT-qPCR. The Relative gene expression levels are in Log 10 scale. * P<0.05.

[0371] FIG. 25 is a graph showing the size distribution of particles secreted by pStemHeps in cell supernatants using nanoparticle tracking analysis (NTA) with the PARTICLEMETRIX ZetaView instrument.

[0372] FIG. 26A-C is a set of schemes and plots showing the detection of tetraspanin on extracellular vesicles (EVs) secreted by pStemHeps by ExoView device. The EVs are first captured on spots by anti-tetraspanin antibodies, then a combination of fluorescent-labelled antibodies against the same tetraspanins are applied and read (Panel A). The results demonstrate particles detection by fluorescence on the four different spots (particles are normalized by the read area, MIgG: isotype control) (Panel B). Finally, the distribution of the number of expressed tetraspanins in all spots are represented (Panel C). The 3 markers are CD63, CD81, and CD9.

[0373] FIG. 27 is a scheme showing the proteomics analysis on cell lysate, cell supernatant, and purified vesicles with a Venn diagram based on the detected proteins (threshold >1 PSM) from three samples.

[0374] FIG. 28 is a plot showing the expression of HGF in the supernatant of a culture of pStemHeps prepared accordingly to the protocol C as measured by ELISA test.

EXAMPLES

[0375] The present disclosure and invention are further illustrated by the following examples. Unless stated otherwise, the term pStemHeps refers herein to the hepatic stem-like cells according to the invention.

Example 1

1.1 Material and Methods

a) Cell Culture

[0376] Human embryonic stem cell (hESCs) lines were derived under current Good Manufacturing Practice (cGMP) conditions on human fibroblast feeder layers and are available in research and clinical-grade formats. hESCs (ESI-BIO) were cultured in feeder-free conditions on culture dishes pre-coated with 5 g/ml Laminin LN521 (BIOLAMINA) in mTeSR1 medium (STEM CELL TECHNOLOGIES) at 37 C. in a 5% CO.sub.2 incubator with daily media changes and were passaged using TrypLE (THERMOFISHER SCIENTIFIC) and then cultured during 24 hours in the presence of 10 M of the Rock inhibitor Y-27632 (STEM CELL TECHNOLOGIES).

b) Hepatic Differentiation In Vitro (Generation of the Population of Hepatic Stem Cells)

[0377] Cells (75,000 cells/cm.sup.2) were plated on laminin LN521 (BIOLAMINA) at 5 g/ml in mTeSR1 (STEM CELL TECHNOLOGIES) containing 10 M of the Rock inhibitor Y-27632 (STEM CELL TECHNOLOGIES). After 24 hours, to start differentiation (Day 0), hESCs maintenance medium was replaced by RPMI supplemented with B27 serum-free supplement (LIFE TECHNOLOGIES) and cells were changed daily thereafter. During the first day of definitive endoderm differentiation induction, cells were cultured in the presence of 100 ng/ml Activin A (MILTENYI BIOTEC), 50 ng/ml Wnt3a (R&D SYSTEMS) and 3 M CHIR-99021 (STEM CELL TECHNOLOGIES). Then cells were cultured for 1 day in the presence of 100 ng/ml Activin A (MILTENYI BIOTEC) and 50 ng/ml Wnt3a (R&D SYSTEMS) and then for 3 days in the presence of 100 ng/ml Activin A (MILTENYI BIOTEC). To induce the hepatic specification after endoderm formation, with 10 ng/ml fibroblast growth factor 10 (FGF-10) (MILTENYI BIOTEC) and 10 ng/ml bone morphogenetic protein 4 (BMP-4) (R&D SYSTEMS) were used for five days. After using this hepatic differentiation protocol A, pStemHeps were frozen in CryoStorm CS10 (STEM CELL TECHNOLOGIES). In order to produce hepatocyte-like cells (HLCs) by hepatic maturation, cells were cultured in hepatocyte culture medium (HCM) (LONZA) supplemented with 20 ng/ml hepatocyte growth factor (HGF) and 20 ng/ml oncostatin M (OSM) (MILTENYI BIOTEC), the media was changed every 2 days.

c) RNA Extraction and Real-Time Quantitative PCR

[0378] Total mRNA was extracted from culture using the RNeasy Mini kit (QIAGEN) following the manufacturer's recommendations. Real-time reverse-transcription was performed starting from 5 ng RNA, with a one-step RT-PCR kit using Taqman technology (AgPath-ID One-Step RT-PCR, LIFE TECHNOLOGIES) and using the Applied Biosystems ViiA 7 Real-Time PCR System and the appropriate primers for Taqman assays (LIFE TECHNOLOGIES): OCT4 (Hs00999632_g1), SOX17 (Hs00751752_s1), HNF4A (Hs00604435-ml), AFP (Hs00173490_ml), and GAPDH (Hs99999905_ml). The relative gene expression was calculated using the 2-ct quantification method after normalization to GAPDH values and expressed as fold of levels found in undifferentiated hESCs cells

d) Flow Cytometry

[0379] Cells were harvested and incubated on ice with Fixable Viability Dye eFluor 450 (eBioscience 65-0863-14) for 20 min. The intracellular staining was carried out according to the manufacturer's instructions using Fixation/Permeabilization kit (eBioscience 00-5123-43 and 00-5223-56) in the presence or absence of primary antibodies against SOX17 (Allophycocyanin Goat anti-SOX17, R&D SYSTEMS, #IC1924A) and HNF4A (Alexa Fluor 488 Mouse anti-HNF4A, SANTACRUZ, #SC-374229). Detection of CXCR4 was performed without cell permeabilization and with anti-CXCR4 monoclonal mouse IgG2b antibody (R&D SYSTEMS, #Mab173) and Alexa Fluor 568 anti-mouse IgG2b. The analysis was performed with a FACSCanto II (BD BIOSCIENCES) and Flow Jo software (TREE STAR, Ashland, OR, USA). Human primary hepatocytes were obtained from Biopredic International.

e) Immunofluorescence Cell Staining Assay

[0380] Cultured cells were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100 in PBS for 15 min and blocked with 1% BSA-0.1% Triton in PBS for 30 min. Primary antibodies were diluted in 11% BSA-0.1% Triton in PBS, and incubated 1 h at room temperature (mouse anti-AFP, SIGMA ALDRICH, #A8452, 1/50; mouse anti-ALB, CEDARLANE, #CL2513A, 1/300; rabbit anti-FOXA2, ABCAM, #ab108422, 1/100; mouse anti-HNF4A, SANTACRUZ, #SC-374229, 1/100; rabbit anti-OCT4, SANTACRUZ, #SC-9081, 1/50). Secondary antibodies were diluted in 1% BSA-0.1% Triton in PBS and incubated for 1 hour at room temperature (Alexa Fluor 488 Donkey anti-mouse IgG, INVITROGEN, #A21202, 1/200; Alexa Fluor 488 Goat anti-rabbit IgG, INVITROGEN, #A21206, 1/200). Cells were mounted using coverslips and ProLong Gold Antifade Mountant (LIFE TECHNOLOGIES). All pictures were observed under a Zeiss fluorescent microscope.

f) Enzyme-Linked Immunosorbent Assay (ELISA) Analysis

[0381] Human AFP and ALB secreted into the culture medium were determined by the Human AFP Elisa Quantitation kit (ABCAM) and the Human Albumin ELISA Quantitation kit (Bethyl; http://www.bethyl.com) following manufacturer's instructions.

[0382] Human AFP secreted into the sera of transplanted animals were specifically determined by the Human AFP Elisa Quantification Kit (EHAFP, THERMOFISHER SCIENTIFIC) following the manufacturer's instructions.

g) Animals and Induction of Acute Liver Failure (ALF)

[0383] Male NOD/SCID mice (6 weeks) were treated with 400 mg acetaminophen (APAP)/kg to induce acute liver failure (ALF) 3 hours prior to cell transplantation. ALF was evaluated by means of histological staining and determination of transaminases in the sera of treated animals. Three hours after the injection of APAP, animals received an intrasplenic injection of 110.sup.6 frozen pStemHeps in 50 L RPMI/B27 medium (LIFE TECHNOLOGIES). All assays were carried out using pStemHeps that had been cryopreserved and thawed. The control mice had received APAP intoxication and no treatment. At 24 h, mice were sacrificed under anesthesia (isoflurane). Blood, serum and liver were collected and stored at 80 C. until analysis. For histological analyses, formalin-fixed livers were dehydrated, embedded in paraffin blocks and cut into 5 m sections. The liver sections were stained with haematoxylin and eosin (H&E), scanned with a digital slide scanner (Nanozoomer S360, Hamamatsu Photonics) at 20 magnification. Digital images were converted to 8-bit grey scale and grey values were measured using ImageJ software to quantitatively evaluate the normal and the necrotic tissue areas. The livers of mice that were not intoxicated with APAP were used to define the grey values of a normal liver.

h) PCR Alu

[0384] Genomic DNA was extracted from tissues using the Genomic DNA from organs and cells Kit (MACHEREY-NAGEL) following the manufacturer's recommendations. Alu PCR is conducted using two primers: hAluR: 5-TTT TTT GAG ACG GAG TCT CGC TC-3 (SEQ ID NO: 1) and hAluF: 5-GGC GCG GTG GCT CAC G-3 (SEQ ID NO: 2). PCR is carried with Herculase Kit (AGILENT) out in a total volume of 25 L with 10 ng of genomic DNA. PCR is carried with Herculase Kit (AGILENT) out in a total volume of 25 L with 10 ng of genomic DNA. PCR running conditions are the ones recommended by the manufacturer.

i) Statistical Analysis

[0385] Data are expressed as mean values SEM. Statistical analysis was made using the GraphPad Prism 7 software (GraphPad Software, San Diego, CA). Statistical significance was assessed using the Student's t test for comparisons between groups. Survival data were analyzed with the Kaplan-Meier test. The statistical significance of the survival rates was determined by a log-rank test. For all tests, P<0.05 was considered significant (*).

1.2 Results

[0386] a) The Differentiation of hESCs cGMP into Hepatic Stem Cells (pStemHeps)

[0387] At day 0 of the differentiation protocol, the hESCs cGMP culture were positive for the pluripotency markers octamer-binding transcription factors 4 (OCT4) (FIGS. 1 and 2A). The hESCs cGMP were subjected to a three steps differentiation protocol. The cells were induced into definitive endoderm (DE), followed by 5 days of hepatic specification where the cells differentiated into hepatic stem-like cells (pStemHeps). FIG. 2A shows the kinetics of expression of keys markers of pluripotency (OCT4), DE (FOXA 2), hepatic progenitors (AFP) and mature hepatocyte (ALB): disappearance of OCT4 expression, expression of FOXA2 after DE induction (Day 5), expression of AFP after hepatic induction (Day 11) and no expression of albumin (ALB). At day 11, pStemHeps expressed AFP, CXCR4 (DE marker), FOXA2, HNF4A and SOX17 (DE marker) as measured by RNA, immunofluorescence and/or cytofluorimetry analyses (FIGS. 1B-D and 2A-C). Accordingly, pStemHeps did secrete AFP (FIG. 2D) but not ALB (FIG. 3C).

[0388] Cryopreservation and thawing procedures have been reported to have detrimental effects on the viability and function of primary human hepatocytes when compared to freshly isolated cells. The successful cryopreservation of pStemHeps successfully retained high viability on a period of two to nine months (Table 1).

TABLE-US-00001 TABLE 1 Cryopreserved and thawing pStemHeps Cell Cell viability Cell viability average viability before before after Cell viability Freezing # freezing freezing freezing average after time Prod (%) (%) (%) freezing (%) (month) 1 94 93 92 91 3 2 91 90 2 3 97 96 9 4 90 85 4
b) The Potency of hESCs to Differentiate into HLCs

[0389] After hepatic maturation of pStemHeps into HLCs, the morphology of the differentiated cells shared many characteristics with adult primary hepatocytes, including a polygonal shape, distinct round nuclei or double nuclei, and numerous vacuoles or vesicles (FIG. 3B). HLCs secreted ALB, an important function displayed by mature hepatocytes (FIG. 3C). Cell immunostaining showed that HLCs were positive for ALB, AFP and HNF4A (FIG. 3A).

c) pStemHeps Rescue from APAP-Induced ALF

[0390] To assess the therapeutic potential of pStemHeps in vivo, the model of acetaminophen toxicity (APAP) in immuno-compromised mice was used, which mimics ALF. This model was first chosen because consistent hepatotoxicity has been shown in murine models and hepatocyte damage in human liver. In western countries, the paracetamol intoxication is the first cause of ALF.

[0391] An acetaminophen dose of 400 mg/kg body weight resulted in a rapid death of control animals as soon as 24 hours after the administration of APAP. pStemHeps transplantation significantly increase animal survival (>90% survival), when compared to control ALF animals (FIG. 4).

[0392] Accordingly, induction of ALF was concomitant with the rapid release of alanine aminotransferase (ALAT, a major liver injury marker) in the blood. pStemHeps transplantation resulted in reduction of ALAT values, as soon as 24 hours after cell therapy, when compared to control ALF animals (FIG. 5).

[0393] Furthermore, histological analysis indicated a higher extent of liver necrosis in control animal group receiving only APAP compared to animal group receiving APAP and pStemHeps within 24 hours post-cell transplantation (FIG. 6), showing a rapid therapeutic benefit of pStemHeps therapy.

[0394] Human AFP was detected in the sera of transplanted mice and not in the sera of control non-transplanted mice at 24 h post cell injection (FIG. 7), showing fast cell recovery and functionality after pStemHep thawing.

[0395] In order to investigate whether the transplanted cells were engrafted within the livers of the recipient mice, the presence of human pStemHeps was detected using human Alu PCR (specific sequence that is highly repeated). Human cells were detected in liver after only 24 h post transplantation (FIG. 8).

1.3 Conclusion

[0396] Cryopreservation and thawing procedures have been reported to have detrimental effects on the viability and function of primary human hepatocytes when compared to freshly isolated cells (Terry et al., 2010). The successful cryopreservation of human hepatic progenitors that retain high viability, as well as the ability to be cultured and further differentiated, would allow for long-term banking of the cells required for subsequent research and clinical applications. The hepatic stem-like cells were able to proliferate and express hepatic specific markers such as HNF4A and AFP. When further maturated, cells showed liver specific functions such as albumin secretion.

[0397] After cell infusion, pStemHeps recover rapidly from cryopreserved state, engrafted and were able to protect mice from lethal acute liver failure. Cells were detected in the liver of the transplanted mice after 24 h. A significant and rapid decrease in serum ALAT and liver tissue necrosis was also reported as compared to controls APAP-ALF mice.

[0398] Here, it is shown that frozen immature hepatocytes produced from human pluripotent stem cells are able to rescue mice from APAP-ALF by accelerating liver regeneration of healthy tissue. pStemHeps rapidly recover and become functional within 24 h post-transplantation. Altogether, these results show that higher regeneration of the healthy liver tissue upon pStemHeps transplantation may thus benefit treatment of fulminant liver failure, such as ALF, and also benefit treatment of fulminant liver failure with preexisting chronic liver diseases, such as ACLF. Indeed, similarly to ALF, ACLF benefits from a regeneration of healthy liver tissue within the diseased liver to treat fulminant liver failure.

Example 2

2.1 Material and Methods

a) Cell Cultures

[0399] Human embryonic stem cell (hESCs) lines were derived under current Good Manufacturing Practice (cGMP) conditions on human fibroblast feeder layers and are available in research and clinical-grade formats. hESCs (ESI-BIO) were cultured in feeder-free conditions on culture dishes pre-coated with 5 g/mL Laminin LN521 (BIOLAMINA) or with 0.5 g/cm.sup.2 vitronectin (GIBCO) in mTeSR1 medium (STEM CELL TECHNOLOGIES) at 37 C. in a 5% CO.sub.2 incubator with daily media changes, and were passaged using TrypLE (THERMOFISHER SCIENTIFIC) and then cultured during 24 hours in the presence of 10 M of the Rock inhibitor Y-27632 (STEM CELL TECHNOLOGIES).

b) Hepatic Differentiation In Vitro and Characterization of the Hepatic Stem Cells

[0400] Cells (20,000 to 50,000 cells/cm.sup.2) were plated on laminin LN521 (BIOLAMINA) at 5 g/ml (protocol A and B) or LN511 (iMatrix 511, AMSBIO) at 0.0625 g/cm.sup.2 (protocol C) cultured in mTeSR1 (STEM CELL TECHNOLOGIES) with daily culture changes, and were passaged using TrypLE (THERMOFISHER SCIENTIFIC) and then cultured during 24 hours in the presence of 10 M of the Rock inhibitor Y-27632 (STEM CELL TECHNOLOGIES).

[0401] After 48 hours, hepatic differentiation of hESCs was then started as performed in example 1 to generate pStemHeps, according to the Table 2, which provides the cultures' protocols.

TABLE-US-00002 TABLE 2 protocols for the preparation of hepatic stem cells from hESCs Protocol number Endoderm induction* Hepatic differentiation* A d0-d1: ACTA + Wnt3A + CHIR-99021 d5-d11: BMP4 + d1-d2: ACTA + Wnt3A FGF10 d2-d5: ACTA (matrix: LN521) (matrix: LN521) B d0-d2: CHIR99021 d3-d8: BMP4 + FGF10 (matrix: LN521) d8-d10: HGF + CHIR99021 (matrix: LN521) C d0-d2: CHIR99021 d3-d8: BMP4 + FGF10 (matrix: iMatrix 511) d8-d10: HGF + CHIR99021 (matrix: iMatrix 511) *Duration of the step with ACT-A (100 ng/ml), Wnt3A (50 ng/ml), CHIR99021 (3 M), BMP4 (10 ng/ml), FGF10 (10 ng/ml), HGF (20 ng/ml).

[0402] The population of hepatic stem-like cells generated by the protocols above were characterized in vitro and in vivo.

[0403] For in vitro characterization, parameters such as the harvested density of cells, the yield, the viability of the cells, the levels of markers for DE specification such as FOXA2 and SOX17, for hepatic stem cell specification such as AFP, APOA1, APOB, HNF1B HNF4A, TBX3, KRT19 and TTR, and for mature hepatocytes specification such as ALB, ASGR1, CYPs F9, NAGS, and UGT1A1 were assessed.

[0404] The level of markers was assessed by real-time RT-PCR, ELISA, FACS, immunofluorescence cell staining as mentioned. Information relative to the qPCR primers for Taqman assays are indicated in example 1 and with qPCR primers (LIFE TECHNOLOGIES) for FOXA2 (Hs00232764_ml), HNF1B primers (Hs01001602-ml), TBX3 (Hs00195612_ml), TTR (Hs00174914-ml). Information relative to the antibodies for immunofluorescence cell staining are indicated in example 1 and with anti-EPCAM (R&D SYSTEMS, #AF960), anti-CK19/KRT19 (DAKO, #M0888), anti-SOX17 (R&D SYSTEMS, #AF1924) anti-KI67 (ABCAM, #Ab15580), and Alexa Fluor 568 Donkey anti-goat IgG (INVITROGEN, #A11057).

[0405] Alternatively, the levels of markers were assessed by mRNA expression profiling by 3 DGE (DGE-Seq). RNA-sequencing protocol was performed on 10 ng of total RNA to determine the number of mRNA molecules per million of total mRNA molecules as described by Kilens et al. (2018).

c) Animals and Induction of Acute Liver Failure (ALF)

[0406] For in vivo characterization, the hepatic stem cells were assessed for their ability to rescue APAP-induced ALF in NOD/SCID mice at a dose of 400 mg/kg or 720 mg/kg (see Example 1 above).

[0407] Alternatively, the NOD/SCID mice with APAP-induced ALF have undergone surgery as to remove approximately of the liver prior to the transplantation with 110.sup.6 cryopreserved pStemHeps, as indicated.

[0408] Alternatively, male C57BL/6 mice (6 weeks) were treated with 1,500 mg thioacetamide (TAA)/kg to induce ALF 24 hours prior to transplantation of 110.sup.6 cryopreserved pStemHeps that was performed as described in example 1.

[0409] At time of sacrifice, blood was collected and serum aliquots were protected from light and stored at 80 C. until analyses measuring human AFP by ELISA as described in example 1.

[0410] The presence of K167 (MKI67)-positive cells was assessed by immunohistochemistry on formalin-fixed/paraffin-embedded liver sections (3 m) at 24 hours after injection of 110.sup.6 pStemHep (prepared as in protocol C; see Table 2) in animals that did received 700 mg/kg body weight of APAP or in untreated control animals that did received APAP only. After paraffin was extracted from sections, endogenous avidin/biotin binding sites were blocked using an Avidin/biotin blocking kit (THERMOFISHER SCIENTIFIC, #00-4303) and endogenous peroxidase activity was inhibited by incubation for 10 minutes in a 3% H.sub.2O.sub.2 solution in PBS. After incubation in Animal Free Blocker (VECTOR LABORATORIES, #SP-5030-250) for 1 hour, rabbit polyclonal anti-KI67 primary antibodies (ABCAM, #ab15580) diluted 1:1,000 in Animal Free Blocker was applied overnight at 4 C. The K167-positive cells were revealed with biotinylated goat anti-rabbit immunoglobulin and streptavidin-peroxidase (ABCAM; #ab64261) using diaminobenzidine as a chromogenic substrate. Slides were counter-stained with hematoxylin and xylene.

d) Statistical Analysis

[0411] Statistical analyses were performed as indicated in example 1.

2.2 Results

[0412] a) The Differentiation of hESCs cGMP into pStemHeps According to 3 Different Protocols

[0413] hESCs have been differentiated in hepatic stem cells accordingly to one among 3 protocols (different matrix and cocktails for hepatic differentiation) as in Table 2. pStemHeps that were produced according to the different protocols all expressed AFP but also FOXA2, HNF1b, HNF4A, TBX3 and TTR as assessed by RTqPCR analyses (FIG. 9). Furthermore, RNAseq analyses of pStemHeps (protocol A and B) showed high expression of the following genes: AFP, APOA1, APOA2, APOB, APOE, CD164, CD24, DPP4, EPCAM, G1A1, GSTA2, KRT18, KRT19, SEPP1, SOD1, SPARC, TTR, VIM, VTN (FIG. 10). They also expressed these genes at a lower relative level: APOA4, BMP2, BMP4, DLKT, GATA4, GATA6, GSTA1, HNF1B, HNF4A, SMAD7, TBX3. And they did not express the following genes characteristic of mature hepatocytes: ABCB11, ASGR1, CYP1A2, CYP2A6, CYP2B6, CYP2B7P, CYP2C9, CYP2E1, CYP3A4, CYP3A7, F9, NAGS, UGT1A1 and did not express PDX1.

[0414] Accordingly, pStemHeps that were produced using Protocol A, B and C did produce and secrete AFP proteins and not ALB proteins (FIGS. 11A and 11B). FIG. 11B also shows that pStemHeps were positive for HNF4A, SOX17, EPCAM, CK19 (KRT19), FOXA2 proteins. pStemHeps were also positive for KI67 proteins, a marker of proliferating cells (FIG. 11B).

b) pStemHeps Rescue from APAP-Induced ALF

[0415] Cryopreserved pStemHeps generated by protocols A, B and C were assessed for their ability to rescue APAP-induced ALF in NOD/SCID mice.

[0416] FIGS. 4, 12, 13A-B and 14A respectively show that the pStemHeps generated by these protocols all promote a significant increase of survival of NOD/SCID mice with acetaminophen-induced ALF (FIG. 4: protocol A, FIG. 12: protocol B, FIGS. 13A-B: protocols B and C, respectively, FIG. 14A: protocol C).

[0417] pStemHeps generated with protocols B and C were able to rescue acetaminophen-induced ALF in NOD/SCID mice having undergone surgery to remove of the liver before transplantation with these cells (see FIG. 13A-B, respectively).

[0418] pStemHeps are able to rescue ALF in NOD/SCID mice intoxicated at a high dose of acetaminophen resulting in 100% mice death within 2 days in untreated control mice group (FIG. 14A).

[0419] As shown in FIG. 14B-C, pStemHeps promote a significant increase of proliferating cells in the liver of NOD/SCID mice with acetaminophen-induced ALF (FIG. 14B) at 24 h post-cell injection as compared to untreated control mice with acetaminophen-induced ALF that did not received pStemHeps (FIG. 14C). These results demonstrate faster liver regeneration after pStemHeps cell therapy in APAP-induced ALF mice.

c) pStemHeps Rescue from Non APAP-Induced ALF

[0420] Cryopreserved pStemHeps generated were assessed for their ability to rescue ALF induced in mice using hepatotoxin thioacetamide (TAA) as an animal model of non-acetaminophen induced human ALF.

[0421] FIG. 15A-B shows that the pStemHeps generated by protocol rescue immunocompetent mice from TAA-induced ALF, in the presence or in the absence of tacrolimus (immunosuppressive agent). Altogether, these results provide evidence about the low immunogenicity of the cells.

[0422] FIG. 16 shows that human AFP was detected in the sera of transplanted mice and not in the sera of control non-transplanted mice at 24 h post cell injection, showing fast cell recovery and functionality after pStemHeps thawing in a non-APAP-induced ALF in mice.

2.3 Conclusions

[0423] Here is shown that the obtention of hepatic stem-like cells (pStemHeps) preparation from hESCs are not limitative steps, since several protocols may be implemented with significantly equivalent therapeutic results to treat acute liver failure in a mouse model. Furthermore, pStemHeps can rescue APAP-induced and non-APAP induced ALF. The pStemHeps became therapeutically active fast enough (within 24 hours) after cell thawing and cell transplantation to rescue mice from ALF (first death occurring within 24 hours) and/or in absence of immunosuppression. Altogether, these results suggest that pStemHeps transplantation allow higher liver regeneration of healthy tissue in conditions of fulminant liver failure, such as ALF, as well as in conditions of fulminant liver failure with preexisting of chronic liver diseases, such as in ACLF. Again, as for ALF, ACLF benefits from a regeneration of healthy liver tissue within the diseased liver to treat fulminant liver failure.

Example 3

3.1 Materials and Methods

[0424] a) Generating Spheroids from pStemHeps

[0425] At the end of the pStemHeps specification differentiation stage (see protocols described in example 2), pStemHeps cells were rinsed with Ca/Mg free PBS (GIBCO) and incubated with TrypLE (LIFE TECHNOLOGIES) for 10 minutes at 37 C. RPMI/B27 (LIFE TECHNOLOGIES) was added to the dissociated cell suspension and cells were gently flushed to be fully dissociated. Cells were then plated into Aggrewell 400 plates (STEM CELL TECHNOLOGIES) to attain a final density of 1,000 cells/spheroid in complete HCM (LONZA) supplemented with 10 M of Y27632 (STEM CELL TECHNOLOGIES), 20 ng/ml HGF (MILTENYI), 20 ng/ml OSM (MILTENYI) and 1 M dexamethasone (SIGMA ALDRICH) (D0 SPHE). Cells were incubated for 48 hours at 37 C., 5% CO.sub.2 with no medium change.

b) Gene Expression Analysis

[0426] The level of markers was assessed by RT-qPCR and ELISA, as mentioned above.

[0427] For immunofluorescence cell staining, spheroids were rinsed with Ca/Mg supplemented PBS and fixed for 30 minutes using 4% PFA, permeabilized with 0.5% Triton in PBS for 15 minutes. Cell Immunostaining were performed by incubating spheroids in PBS containing 0.1% Triton and 1% BSA (blocking buffer) for 1 h with the primary antibodies, and 1 h with the appropriate secondary antibodies at room temperature (Table 3).

TABLE-US-00003 TABLE 3 Antibodies used for immunofluorescent staining of spheroids Target Species Provider's Reference Dilution Primary antibodies ALB ms CEDARLANER #CL2513A 1/300 CK19 ms DAKOR #M0888 1/100 AFP ms SIGMA ALDRICH #A8452 1/50 HNF4A ms SANTACRUZ #SC-374229 1/100 FOXA2 rbt ABCAM # AB108422 1/100 SOX17 gt R&D SYSTEMS #AF1924 1/40 Secondary antibodies Dk anti-ms AF488 INVITROGEN #A21202 1/200 Gt anti-rbt AF568 INVITROGEN #A11036 1/200 Dk anti-gt AF568 INVITROGEN #A11057 1/200

b) Cell Viability

[0428] To assess in vitro cell viability, 0.4 M calcein-AM and 4 M ethidium homodimer-1 (LIVE/DEAD viability/cytotoxicity kit, LIFE TECHNOLOGIES) and 10 g/ml Hoechst 33342 (LIFE TECHNOLOGIES) were added to the culture medium for 30 minutes before imaging.

c) Transplantation of Spheroids and In Vivo Functionality

[0429] Before transplantation, spheroids were embedded in alginate hydrogels. For this, two-days cultured spheroids were generated as described above, harvested from Aggrewell, centrifuged at 100g for 5 min and resuspended in calcium/magnesium free PBS (LIFE TECHNOLOGIES). Spheroids were mixed with 8.9% ultra-pure sodium alginate, with low viscosity and high glucoronic acid (Pronova SLG20, NOVAMATRIX) and then gently mixed with 0.0225 M CaCO.sub.3 (SIGMA ALDRICH), and 0.045 M glucono-d-lactone (SIGMA ALDRICH) to attain a final cell concentration of 2010.sup.6/ml (approximately 210.sup.4 spheroids). Gelation of 250 l hydrogels took place at room temperature for 3 min. Two 250 l pre-molded alginate hydrogel containing or not spheroids were intraperitoneally transplanted under laparotomy into immunocompetent C57BL/6.

[0430] To assess in vivo viability of spheroids, alginate hydrogels were harvested from transplanted animals at day 8 post-transplantation, and dissociated using a solution of PBS without calcium and magnesium (LIFE TECHNOLOGIES) containing 0.1 M EDTA (LIFE TECHNOLOGIES), and 0.2 M sodium citrate tribasic (SIGMA ALDRICH). After complete hydrogel dissociation, spheroids were spun down at 100g for 5 min, incubated in fresh RPMI/B27 containing 0.4 M of calcein-AM and 4 M ethidium homodimer-1 (LIVE/DEAD viability/cytotoxicity, LIFE TECHNOLOGIES) and 10 g/ml Hoechst 33342 (LIFE TECHNOLOGIES) for 90 minutes before imaging.

[0431] Gene expression levels in spheroids was assessed at 8 days post-transplantation by real-time RT-PCR after harvesting and dissociating alginate hydrogels as described above. Serum AFP of transplanted animals were determined by the AFP Elisa Quantification Kit specific for human AFP (EHAFP, THERMOFISHER SCIENTIFIC) following the manufacturer's instructions.

d) Statistical Analysis

[0432] Statistical analyses were performed as indicated in example 1

3.2 Results

[0433] As shown in FIG. 17A-C either freshly-prepared or cryopreserved pStemHeps could aggregate and form spheroids in 48 hours after plating into Aggrewell plates. At harvest, spheroids had high esterase activity and were not positive for ethidium homodimer-1 staining. Only non-aggregated cells were positive to ethidium homodimer-1 and did not show any esterase activity. These results showed high viability of all spheroids. In addition, spheroids prepared from freshly-prepared or cryopreserved pStemHeps expressed high levels of AFP, HNF4A and SOX17 mRNA, similar to those of pStemHeps from which they were prepared (FIG. 18).

[0434] An immunofluorescent assay was further performed on spheroids, in order to assess the levels of expression of some key markers of hepatic differentiation. Results showed that spheroids express AFP, CK19 (KRT19), FOXA2, HNF4A, and SOX17 proteins, whereas they did not express the ALB proteins (FIG. 19).

[0435] Finally, ELISA for AFP performed on cell supernatant samples confirmed that spheroids secreted AFP, which level was similar level to that of pStemHeps (FIG. 20A). In addition, no secretion of ALB by the 2 days-cultured spheroids was found, in contrast to HLCs (FIG. 20B).

[0436] To evaluate the in vivo functionality of spheroids, two pre-molded alginate hydrogels containing a total of about 1010.sup.6 pStemHeps were intraperitoneally transplanted in CB57BL/6 mice. In addition, 210.sup.7 pStemHeps were intraperitoneally transplanted in CB57BL/6 ALF-mice and rescue the survival (FIG. 21).

[0437] FIG. 22 shows that human AFP was detected in the sera of C57BL/6 mice at 2 days post-transplantation with pre-molded alginate hydrogels containing spheroids at a mean level of about 320 ng/ml.

[0438] After 8 days post-transplantation, spheroids embedded in alginate hydrogels are highly viable (FIG. 23) and expressed AFP and HNF4A at a similar level than that of spheroids (non-embedded in alginate hydrogel) cultured in vitro during 8 days (FIG. 24A-B).

3.3 Conclusions

[0439] Here is shown that it is possible to quickly and efficiently generate spheroids from either freshly-prepared or cryopreserved isolated pStemHeps cells. Spheroids show high viability in vitro and maintain their progenitor status as similar to pStemHeps cells, in particular with expression of the AFP marker and no expression of the ALB marker. These spheroids, which were embedded in alginate hydrogels, have the ability to secrete AFP in vivo and survive for at least 8 days in the peritoneal cavity of immune competent animals, which is a time sufficient enough to rescue mice from acute liver failure (death occurs within 5 days, see example 1 and example 2). In addition, pStemHeps transplanted in the in peritoneal cavity rescue mice from ALF. Altogether, these results support that pStemHeps embedded in hydrogels may thus benefit treatment of fulminant liver failure, such as ALF, and also benefit treatment of fulminant liver failure with preexisting chronic liver diseases, such as ACLF.

Example 4

4.1 Materials and Methods

[0440] a) Purification and Size Distribution of Extracellular Vesicles (EVs) Secreted by pStemHep in Cell Supernatants

[0441] Cell supernatant was clarified at 2,000g during 10 min to remove cellular debris. Then it was aliquoted in 40 ml tubes and frozen at 80 C. Three tubes were thawed, and particles size distribution and concentration were determined by nanoparticle tracking analysis (NTA) using a ZetaView (PARTICLEMETRIX, Germany) with a 405 nm laser. Before measurements, EVs were diluted 100 times with sterile PBS (confirmed to be particle-free by NTA measurement). For each sample, a sensitivity of 80 and a shutter of 100 were set.

b) Further Characterization of EVs

[0442] EVs from cell supernatant were concentrated and purified by two consecutive runs of ultracentrifugation at 150,000g during 90 min using an optima MAX-XP ultracentrifuge (BECKMAN COULTER, UK). Concentrated EVs were analyzed by ExoView (NANOVIEW BIOSCIENCES, USA). The sample was diluted at 110.sup.8 EV/ml in the kit's reagent A. The sample was incubated on the ExoView Tetraspanin Chip for human EVs placed in a 24-well plate for 16 h at room temperature. The chips were washed 3 times with reagent A. Chips were incubated with ExoView Tetraspanin Labelling antibodies that consist of anti-CD81 Alexa-555, anti-CD63 Alexa-488, and anti-CD9 Alexa-647. The antibodies were diluted 1:600 in immunofluorescence blocking solution. The chips were incubated with 250 l of the labelling solution for 1 h, washed in solution A (PBS with 0.05% Tween-20), then in solution B (PBS alone) 3 times and dried. The chips were imaged with the ExoView R100 reader using the NScan acquisition software. The data were analyzed using ExoViewer.

[0443] Human HGF was specifically determined by the Human HGF Elisa Quantification Kit (THERMOFISHER SCIENTIFIC) following the manufacturer's instructions.

c) NanoLC-MS/MS Protein Identification and Quantification

[0444] S-Trap micro spin column (PROTIFI, Hutington, USA) digestion was performed on 40 g of cell lysate, supernatant and extra-vesicles according to manufacturer's instructions. Briefly, proteins were alkylated with 50 mM iodoacetamide in 5% SDS and 1.2% aqueous phosphoric acid. Colloidal protein particulate was formed with the addition of 6 times the sample volume of S-Trap binding buffer (90% aqueous methanol, 100 mM TEAB, pH 7.1). The protein mixtures were transferred into the S-Trap micro columns and centrifuged at 4,000g for 30 seconds, and washed with 150 L S-Trap binding buffer. Samples were then digested with 4 g of trypsin (PROMEGA) at 47 C. for 1 h. Peptides were eluted according to the manufacturer's protocol, and dried in Speed Vacuum.

[0445] They were resuspended in 10% ACN, 0.10% TFA in HPLC-grade water for LC-MS+MS analysis using the nanoRSLC-Q Exactive PLUS (RSLC Ultimate 3000 (THERMOFISHER SCIENTIFIC, Waltham MA, USA). Peptides (1-2 g) were loaded onto a p-precolumn (Acclaim PepMap 100 C18, cartridge, 300 m i.d.5 mm, 5 m, THERMOFISHER SCIENTIFIC), and were separated on a 50 cm reversed-phase liquid chromatographic column (0.075 mm ID, Acclaim PepMap 100, C18, 2 m, THERMOFISHER SCIENTIFIC) using mobile phase A (H.sub.2O with 0.1% formic acid), and mobile phase B (80% acetonitrile, 0.08% formic acid). Peptides were eluted from the column with a gradient of 5% to 40% for 120 minutes, of 40% to 80% for 1 minute, and then the gradient stayed at 80% for 5 minutes after which it returned to 5% to re-equilibrate the column for 20 minutes before the next injection.

[0446] Eluted peptides were analyzed by data dependent MS/MS, using top-10 acquisition method and were fragmented by higher-energy collisional dissociation (HCD). MS scans and MS/MS scans were performed at a resolution of 70,000 and 17,5000 respectively. MS and MS/MS AGC target were set to 310.sup.6 and 110.sup.5 counts with maximum injection time set to 200 ms and 120 ms, respectively. The MS scan range was from 400 to 2,000 m/z. Dynamic exclusion was set at 30 seconds.

[0447] The MS files were processed with the Proteome Discoverer software version 2.4.0.305 and searched with Mascot search engine against the UniProtKB/Swiss-Prot Homo sapiens database (release 15 Apr. 2019, 20415 entries). To search parent mass and fragment ions, a mass deviation was set to 3 ppm and 20 ppm respectively. Other search parameters included: a minimum peptide length of 7 amino acids with a strict specificity for trypsin cleavage, carbamidomethylation (Cys) as fixed modification, whereas oxidation (Met) and N-term acetylation as variable modifications.

4.2 Results

[0448] a) Purification and Size Distribution of EVs Secreted by pStemHep in Cell Supernatants

[0449] Cell supernatant was studied by NTA. A high number of particles of 4.20.410.sup.9 particles/mL was measured, which corresponds to a total particles of (2.00.2)10.sup.12 for 210.sup.9 seeded cells, meaning around 10.sup.3 particles/cells. Their size around 100 nm corresponds well to an EVs distribution (FIG. 25 and Table 4).

TABLE-US-00004 TABLE 4 EVs size distribution Mean.sup.1 Diameter mode (nm) 98.0 5.5 Diameter Mean (nm) 103.9 10.9 Concentration (particle/mL) 4.2 0.4 E+09 .sup.1diameter mean, mode, and concentration of particles present in supernatant samples, represented as the mean SD of three independent measurements by NTA.

b) Further Characterization of the EVs

[0450] The obtained data indicated that EVs could be immuno-captured by the three tested anti-tetraspanins (CD63/CD81/CD9 antibodies; see FIG. 26A) with around 150 to 300 particles detected per spot. Most of the signal was associated with CD63 (40-70%) and to a lesser extent with CD81 markers (35-70%) (FIG. 26B). Respectively 2 and 16% of the immuno-captured EVs were expressing 3 or 2 of the 3 tetraspanin markers (FIG. 26C). This confirms the presence of EVs (tetraspanin positive) among the purified particles.

c) NanoLC-MS/MS Protein Identification and Quantification

[0451] Proteomic analysis on the purified vesicles was compared with proteins from the cell lysate and of the whole supernatant. From the 0,5 g of proteins analyzed, the vesicle sample showed the highest diversity in expressed proteins, with most of them shared with the cell lysate (FIG. 27). Many cytosolic and membrane proteins classically present in EVs were encountered in the vesicle sample at a high concentration. With this proteomic analysis, it was confirmed that there was significant EVs and exosomes in the supernatant of the cultured cells and that it was possible to purify them. Of note, EVs contains the same proteins of interest as the cells, such as AFP, HGF, apolipoteins (Tables 5 and 6, FIG. 28), as well as proteins that are usually found in EVs (Table 7).

TABLE-US-00005 TABLE 5 Proteins of interest that are found in the EVs Uniport Purified accession Protein description (Gene name) vesicles P02771 Alpha-fetoprotein O (AFP) 3 P02647 Apolipoprotein A-I (APOA1) 20 P04114 Apolipoprotein B-100 (APOB) 11 P02649 Apolipoprotein E (APOE) 45 P27487 Dipeptidyl peptidase 4 (DPP4) 49 P17302 Gap junction alpha-1 protein (GJA1) 2 P14210 Hepatocyte growth factor (HGF) 2 P05783 Keratin, type I cytoskeletal 18 (KRT18) 23 P08727 Keratin, type I cytoskeletal 19 (KRT19) 24 P09486 SPARC 2 P08670 Vimentin O (VIM) 42

TABLE-US-00006 TABLE 6 Other proteins of interest that are found in EVs Uniport Purified accession Protein description (Gene name) vesicles P07585 Decorin (DCN) 14 P51654 Glypican-3 (GPC3) 6 P17936 Insulin-like growth factor-binding protein 3 (IGFBP3) 11 P23229 Integrin alpha-6 (ITGA6) 14 P40189 Interleukin-6 receptor subunit beta (IL6ST) 5 P13796 Plastin-2 (LCP1) 11

TABLE-US-00007 TABLE 7 Proteins that are usually found in EVs Uniprot Purified accession Protein description (Gene name) vesicles P60709 Actin, cytoplasmic 1 (ACTB) .sup.(1) (2) 99 P63261 Actin, cytoplasmic 2 (ACTG1) .sup.(1) (2) 98 P06733 Alpha-enolase (ENO1) .sup.(1) 56 P07355 Annexin A2 (ANXA2) .sup.(1) (2) 53 P08758 Annexin A5 (ANXA5) .sup.(1) (2) 41 P08133 Annexin A6 (ANXA6) .sup.(2) 75 P98160 Basement membrane-specific heparan sulfate 52 proteoglycan core protein (HSPG2) .sup.(3) Q00610 Clathrin heavy chain 1 (CLTC) .sup.(2) 99 P68104 Elongation factor 1-alpha 1 (EEF1A1) .sup.(1) 40 P13639 Elongation factor 2 (EEF2) .sup.(1) 22 P04406 Glyceraldehyde-3-phosphate dehydrogenase 68 (GAPDH) .sup.(1) P11142 Heat shock cognate 71 kDa protein (HSPA8) .sup.(1) (2) 47 P07900 Heat shock protein HSP 90-alpha (HSP90AA1) .sup.(1) (2) 43 P08238 Heat shock protein HSP 90-beta (HSP90AB1) .sup.(1) (2) 54 P14618 Pyruvate kinase PKM (PKM) .sup.(1) 35 P02786 Transferrin receptor protein 1 (TFRC) .sup.(3) 21 Q71U36 Tubulin alpha-1A chain (TUBA1A) .sup.(2) 46 P68363 Tubulin alpha-1B chain (TUBA1B) .sup.(2) 49 P07437 Tubulin beta chain (TUBB) .sup.(2) 48 Q13885 Tubulin beta-2A chain (TUBB2A) .sup.(2) 47 Q9BVA1 Tubulin beta-2B chain (TUBB2B) .sup.(2) 47 Q13509 Tubulin beta-3 chain (TUBB3) .sup.(2) 27 P04350 Tubulin beta-4A chain (TUBB4A) .sup.(2) 36 P68371 Tubulin beta-4B chain (TUBB4B) .sup.(2) 45 Q9BUF5 Tubulin beta-6 chain (TUBB6) .sup.(2) 27 .sup.(1) Proteins from the top twenty highest occurring proteins found in studies on EVs (ExoCarta database), .sup.(2) Cytosolic proteins recovered in EVs (MISEC 2018 classification); .sup.(3) the transmembrane or GPI-anchored proteins associated to plasma membrane and/or endosomes.

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