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WO2008035169A2 - Procédé de préparation de cellules hépathiques à partir de cellules progénitrices présentes dans le sang du cordon ombilical - Google Patents

Procédé de préparation de cellules hépathiques à partir de cellules progénitrices présentes dans le sang du cordon ombilical Download PDF

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WO2008035169A2
WO2008035169A2 PCT/IB2007/002699 IB2007002699W WO2008035169A2 WO 2008035169 A2 WO2008035169 A2 WO 2008035169A2 IB 2007002699 W IB2007002699 W IB 2007002699W WO 2008035169 A2 WO2008035169 A2 WO 2008035169A2
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cells
process according
hepatic
preparation
cell
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WO2008035169A3 (fr
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Annalisa Crema
Cristina Haas
Massimo Sanchez
Antonella Lisi
Antonio Ponzetto
Ercole Brunetti
Rodolfo Marchese
Guido Carloni
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Universita' Degli Studi Di Torino
Consiglio Nazionale Delle Ricerche
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells

Definitions

  • This technique presents the disadvantage that the transplanted heterologous hepatic cells are subjected to a violent immune rejection reaction (Susick 2001).
  • adult stem cells are found to be particularly promising, including so called “oval cells” which can be activated to proliferate and differentiate into cells with mature hepatic phenotype (Shafritz & Dabeva, 2002) though their activation, differentiation and proliferation mechanisms are still very incomplete.
  • hepatic progenitors have not been defined with certainty, whereas the main markers of differentiated hepatocyte functionality are well known: production of albumin, urea, glycogen, cytokeratin-18, etc.
  • Oval cells for example express some hepatocyte and bile ducts markers (cytokeratin-7 and -19) and also embryonic hematopoietic cell markers, such as: Thy1 , CD34, CD45, SCA1 , c-Kit and Flt-3.
  • hepatocyte precursors is limited both in terms of reproducibility and the histocompatibility problems in non-autologous transplants.
  • PASC pluripotent adult stem cells
  • hepatocytes have been obtained in vivo (Lagasse et al., 2000) or in vitro (Schwartz 2002) not only from bone marrow cells but also from cells isolated from peripheral blood (PBMCs) (Zhao 2003).
  • PBMCs peripheral blood
  • umbilical cord cells from which both hematopoietic stem cells (HSCs) (Nakahata 1982) and mesenchymal stem cells (MSCs) (Huss 2000) can be extracted.
  • HSCs hematopoietic stem cells
  • MSCs mesenchymal stem cells
  • Cord derived hematopoietic stem cells are as clonogenic as stem cells derived from bone marrow or from adult stem cells, responding to various growth factors and possessing the capacity to expand in vitro in long-term cultures (De Wynter 1998). It has been shown that umbilical cord derived stem cells are useful and effective substitutes for cells derived from bone marrow in allogeneic transplants during the course of therapy for malignant hemopathies.
  • Umbilical cord cells also exhibit some embryonic characteristics (McGuckin 2005) which render them particularly interesting: they have a great potential for ex vivo expansion, are more immunotolerant and have longer telomeres than other adult stem cells. For these reasons cord derived cells are particularly indicated as a source of stem cells for cell transplant therapies in serious hepatic insufficiencies (Kakinuma 2003). The possibility of obtaining phenotypically pure progenitor populations which, once expanded, can be suitably cultivated and directed into specific differentiation lineages, is a further aspect of cord derived stem cells which can facilitate their experimental and therapeutic usage.
  • medulla derived HSCs can give rise mainly to lymphoid, myeloid and myocytic cell lines, but also to cardiac and neuronal cell lines.
  • the mesenchymal cells present in cord blood are cells capable of prolonged proliferative activity having the substrate adhesion characteristics associated with a unique surface molecule expression profile (Lee 2004).
  • BM bone marrow
  • the present invention concerns a process for obtaining hepatic cells in culture starting from a homogeneous population of CD133 + /CD34 + precursors purified from human umbilical cord blood (UCB), which can be amplified while maintaining the same stem cell characteristics and the regenerative potential of the initial population.
  • UMB umbilical cord blood
  • the present invention concerns a process for obtaining cultures of differentiated hepatocytes starting from progenitor cells present in the umbilical cord blood of normal newborns.
  • the invention relates to a process for obtaining a long-term culturing system which allows the in vitro expansion and differentiation of purified hepatic precursors.
  • Pluripotent progenitor cells are purified by immuno- selection, for example with magnetic beads, and expanded in vitro starting from mononuclear cells present in umbilical cord blood.
  • the present invention also relates to optimum growth conditions for achieving hepatocyte commitment.
  • the hepatocyte culture obtained with the presently identified system is suitable for cell therapy replacing diseased hepatocytes with healthy hepatic cells deriving from homologous stem cells.
  • This hepatocyte culture can also be used for the development of bioartificial organs and, in a further application, for the preparation of in vitro systems for the in vitro production of hepatotropic viruses and/or of their components (for example the HCV virus).
  • Figure 1 Growth curve of purified CD133 + /CD34 + cell population in 3 weeks of culturing.
  • Figure 2. Phase-contrast photograph of selected CD133 + mononuclear cells (MNCs) at 9 days from the beginning of treatment with inducers of hepatocyte commitment. The cells become adherent to the substrate and elongated fibroblastoid forms are initially observed.
  • Figure 3. Phase contrast micro-photograph of selected CD133 + MNCs at 13 days from the start of treatment with inducers of hepatocyte commitment. The cells assume stellate form.
  • Figure 4. Phase contrast micro-photograph of selected CD133 + MNCs at 35 days from the start of treatment with inducers of hepatocyte commitment.
  • Figure 6. Kinetics of release and accumulation of albumin and urea in culture medium (immunoenzymatic assays) in response to treatment with the hepatic induction cocktail.
  • Figure 7. Western-blot of the main proteins of hepatic synthesis in the cell supernatant. By means of specific antibodies, the presence of albumin and alpha- fetoprotein was detected together with their accumulation in the supernatant after induction, concurrent with the morphological changes upon differentiation. The days following induction are indicated in the figure above each lane (d ⁇ , d27, d34).
  • CD133 + cells derived from umbilical cord blood can be directed, by suitable conditions of growth and amplification, towards the hepatocytic lineage; they constitute a progenitor line which, with its high proliferative capacity, gives rise to cells expressing hepatic markers. It was hitherto known that neuronal and myocyte cells, and cardiomyocytes, could be derived from umbilical cord blood cells, but a homogeneous population of hepatic precursors with long-term stability has never been obtained.
  • the in vitro process for obtaining hepatic cells from UCB-derived cells is divided into the following steps: a) purification of CD133 + precursors present among umbilical cord blood nucleated cells; b) in vitro expansion of said cells; c) induction of commitment and hepatocyte differentiation; d) obtaining a long-term culture from said cells suitable for therapeutic use.
  • the selection of CD133 + precursors in step a) is preferably carried out after purification of nucleated cells from umbilical cord blood by means of a density gradient, such as Ficoll-Hypaque, in accordance with known methods.
  • the selection of CD133 + ceils is preferably carried out by immunoaffinity and is based on the use of specific antibodies.
  • said antibodies are bound to a solid phase, for example magnetic beads, by i) positive immunomagnetic selection of CD133 + cells, ii) magnetic separation preferably on Miltenyi columns, iii) elution of the CD133 + cell population.
  • the purified CD33 + cells are found to be over 99% pure. Expansion of the purified CD133 + cell population preferably takes place in Iscove culture medium containing 20 ng/ml of Stem Cell Factor (SCF), with added 15- 30% fetal bovine serum (Bio Whittaker Australian FBS, Cambrex). Seeding takes place in accordance with known methods: preferably at an initial concentration of 0.25 x 10 6 cells/ml.
  • SCF Stem Cell Factor
  • the expansion period is preferably between 5 and 15 days.
  • CD133 + cells are obtained which are found to express, after immunophenotypization, the same cell surface antigens as hepatic stem cells, thus confirming their potential to differentiate into the hepatocyte line.
  • said cells are found to be positive for CD133 as expected on the basis of the selection carried out, and also for CD33, CD34, CD43, CD44 but negative for CD90, CD10 and CD2, as indicated by immunophenotyping with specific antibodies.
  • Induction to differentiate into the hepatocytic line c) is carried out in a culture medium, preferably EDM (Eagle's Dulbecco Modified) comprising two or more of the following growth and differentiation factors: FGF- ⁇ , FGF- ⁇ , HGF.
  • EDM Eagle's Dulbecco Modified
  • the concentration of FGF ( ⁇ and ⁇ ) is preferably between 5 and 10 ng/ml, and that of HGF between 10 and 40 ng/ml.
  • the medium also comprises preferably 5 to 10 ng/ml of the following components: EGF (Epidermal Growth Factor), LIF (Leukemia Inhibitory Factor), SCF (Stem Cell Factor), oncostatin-M (OSM) and the Clonetics Singe Quots mixture (CAMBREX) which contains insulin, transferrin, ascorbic acid according to the information given by the supplier, and preferably about 1 ⁇ M of hydrocortisone. Treatment with the induction cocktail is repeated each time the medium is changed and for at least 1 month but preferably for at least 7 weeks.
  • EGF Epidermatitis
  • LIF Leukemia Inhibitory Factor
  • SCF Stem Cell Factor
  • OSM oncostatin-M
  • CAMBREX Clonetics Singe Quots mixture
  • Treatment with the induction cocktail is repeated each time the medium is changed and for at least 1 month but preferably for at least 7 weeks.
  • This treatment induces a characteristic morphological change in the selected population which is well documented in figures 2-5: firstly into elongated fibroblastoid forms and stellate forms (7-10 days), then into bi-and poly-nuclear cells (from 13 to 14 days) with a substantial increase in cell volume typical of the mature hepatocyte, and a distinct increase in the presence of roundish cells.
  • expression of specific hepatic markers is observed such as: alpha- fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18) and cytokeratin 19 (CK19). These markers are found to be positive only after treatment with the mixture of hepatic commitment inducers protracted over a few days or preferably for at least 120 hours or even more preferably for at least 1 week or for at least 5 weeks.
  • the expression of specific hepatic markers continues for at least 7 weeks in the CD133 + cell population cultivated in Dulbecco medium, indicating that the obtained cells stably maintain their commitment to the hepatic line.
  • a more thorough analysis of the experimental data in table 3 shows that it is actually the persistent expression of AFP after 5-7 weeks' induction which is indicative of said commitment, since AFP expression decreases then stops in cells which differentiate into the biliary line (cholangiocytes).
  • the method of the present invention overcomes the reproducibility and efficiency problem of the response to the differentiation stimulus in that, under the selective conditions described, the cell population is qualitatively and quantitatively more homogeneous than that obtained with known methods in the prior art.
  • the population obtained from UCB MNCs with the selection and differentiation method established in the present invention shows a higher ex vivo expansion potential than that obtainable with unselected mononuclear cells (MNCs), therefore allowing therapeutically useful quantities of cells to be obtained more easily, a lower rate of apoptosis, less donor risk and less incidence of graft- versus-host disease (GVHD).
  • MNCs mononuclear cells
  • GVHD graft- versus-host disease
  • the CD133 + /CD34 + subclass of adherent primitive stem cells has a higher GM-CSF production than that of non-adherent cultures. This factor is implicated in the mobilization of stem cells and plays a fundamental role in cellular regeneration processes.
  • the invention relates to the use of the described process for preparing cell compositions with high regenerative potential for use in the hepatic regeneration required following cirrhosis, hepatectomy, post- trauma hepatic damage and hereditary or acquired hepatic pathologies, for example of viral etiology.
  • the method of the invention and the cells obtained therefrom are hence of therapeutic use in treating the damage caused by the aforesaid hepatic pathologies: cirrhosis, hepatectomy, post-trauma hepatic damage and hereditary or acquired hepatic pathologies, for example of viral etiology, by the administration of compositions of cells committed in vitro in accordance with the process of the invention, and administered in useful quantities to patients by infusion, injection or other known methods.
  • the main embodiment of the hepatocyte culture obtained with the thus identified system is cell therapy which replaces diseased hepatocytes with healthy hepatic cells deriving from homologous stem cells.
  • hepatocyte culture also include the development of bioartificial organs, and, in a further embodiment, the preparation of in vitro systems for the in vitro production of hepatotropic viruses and/or their components (for example HCV and HBV viruses etc).
  • Example 1 Enrichment of CD133 + cells from umbilical cord blood
  • the blood (around 150 ml) obtained from a single umbilical cord was diluted 1 :4 with culture medium, then stratified on Ficoll-Hypaque in a 2:1 ratio and centrifuged for 30 minutes at 1800 rpm at ambient temperature. About 2/3 of the supernatant was removed by aspiration.
  • the interface between the supernatant and the Ficoll-Hypaque, containing the mononuclear cells (MNCs) was collected by circular aspiration, washed twice with medium and once with PBS containing added 0.1% BSA and 2 mM EDTA.
  • a batch of the mononuclear cells thus obtained was used for antigenic typing of surface markers.
  • MNCs mononuclear cells obtained (about 8 x 10 8 cells) were incubated with microbeads conjugated to anti-CD133 antibodies and subjected to positive immunomagnetic selection on small columns (Miltenyi, MACS Separation
  • the purified CDT33 + cell population was placed in ISCOVE culture medium containing 20 ng/ml of Stem Cell Factor (SCF), at an initial concentration of 0.25 x
  • Figure 1 clearly shows the high capacity for in vitro expansion of said cells, with a 160-fold increase in cell numbers in 3 weeks of culturing.
  • Table 2 shows that the expression profile of the cell surface markers of CD133 + /CD34 + cells after 14 days of in vitro expansion is analogous to that of hepatic stem cells obtained starting from fetal livers (Barcena et al. 1995) confirming the hypothesized presence of hepatic precursors in the selected cell population.
  • Antigenic typing of the selected and enriched cells was carried out by cytofluorometry.
  • Both the MNCs and the purified CD133 + cells were incubated for 30 minutes at 4°C with pretitrated saturating solutions of the following monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC): anti-CD133 (PE Miltenyi); anti-CD34 (APC Miltenyi); anti- CD7; anti-CD45 and anti-CD90 (FITC Immunotools); anti-OV6 (indirect with secondary FITC rat anti-mouse) (Table 1).
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • APC allophycocyanin
  • anti-CD133 PE Miltenyi
  • anti-CD34 APC Miltenyi
  • anti- CD7 anti-CD45 and anti-CD90
  • FITC Immunotools anti-OV6 (indirect with secondary FITC rat anti-mouse) (Table 1).
  • the CD133 + cells expanded in culture for 2 weeks were incubated with antibodies against the principal surface markers representative of the various differentiated cell lineages and/or of stem cells.
  • APC anti-CD133
  • APC anti-CD34
  • CD43 anti-CD44
  • anti-CD10 anti-CD2
  • anti-CD33 anti-CD90
  • antic-Kit all FITC
  • Example 4 Induction of differentiation into the hepatocyte line After expansion in culture the pluripotent stem cells were induced towards hepatocyte differentiation by incubation in a medium comprising a mixture of specific growth factors.
  • the expansion medium ISCOVE
  • DME Dulbecco's Modified Eagles Minimum Essential Medium
  • BIT 9500 serum substitute Stem Cell Technology
  • fetal serum with added growth factors and hepato-specific hormones in suitable concentrations.
  • the induction mixture contained the following as growth factors: EGF 10 ng/ml; FGF ⁇ 10 ng/ml; FGF ⁇ 10 ng/ml; LIF 10 ng/ml; OSM 10 ng/ml; HGF 15-50 ng/ml and SCF 10-30 ng/ml (Immunotools); Insulin hormones, transferrin and ascorbic acid contained in Clonetics HCM Single Quots (CAMBREX), in accordance with the producer's instructions, and hydrocortisone 1 ⁇ M (SIGMA).
  • the cells were seeded onto 4 chamber slides (LAB-TEK) coated with 0.1% gelatin at an initial concentration of 0.4 x 10 6 cells/ml and analysed for expression of hepatic markers.
  • Figures 2-5 clearly indicate that following administration of the hepato-specific induction cocktail, the cells change their morphology. They become adherent to the substrate and, initially, elongated fibroblastoid (Fig 2) and stellate (Fig 3) forms are observed then, later, bi- and polynuclear cells with a substantial increase in cell volume as is typical of the mature hepatocyte, with a distinct increase in the presence of roundish cells which over time evolve into parenchymal cells similar to mature hepatocytes (Figs. 4 and 5).
  • Table 3 shows that, as well as the morphological changes in the aforedescribed cells, a significant increase in positiveness for expression of hepatocyte-specific markers including Albumin (AIb), Alpha-fetoprotein (AFP), Cytokeratin-18 and 19 (CK18, CK19) were found.
  • AIb Albumin
  • AFP Alpha-fetoprotein
  • Cytokeratin-18 and 19 CK18, CK19
  • Figure 6 documents the release and accumulation in culture medium of albumin and urea, their being the principle products of hepatic synthesis which according to the enzymatic assays carried out, start being detected in the medium at around 8 days after induction.
  • the production of proteins and products of the hepatic line was also confirmed by the "western blot" method in the cell supernatant (Fig. 7) which clearly shows the presence of bands specific for albumin and alpha- fetoprotein as well as their increase over time.

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Abstract

La présente invention concerne un procédé d'obtention de cultures d'hépatocytes différenciés à partir de cellules progénitrices présentes dans le sang de cordon ombilical de nouveau-nés normaux. En particulier, l'invention concerne un procédé d'obtention d'un système de culture de longue durée qui permet l'expansion et la différenciation in vitro de précurseurs hépatiques purifiés. Des cellules progénitrices pluripotentes sont purifiées par immuno-sélection, par exemple, avec des billes magnétiques et développées in vitro à partir de cellules mononucléaires présentes dans le sang de cordon ombilical. La culture d'hépatocytes obtenue par le système ainsi identifié est appropriée pour une thérapie cellulaire qui remplace des hépatocytes malades par des hépatocytes sains issus de cellules souches homologues. Cette culture d'hépatocytes peut également être utilisée pour le développement d'organes bio-artificiels et, dans un autre mode de réalisation, pour la préparation de systèmes in vitro pour la production in vitro de virus hépatotropes et/ou de leurs composants (à savoir, le virus VHC).
PCT/IB2007/002699 2006-09-18 2007-09-18 Procédé de préparation de cellules hépathiques à partir de cellules progénitrices présentes dans le sang du cordon ombilical WO2008035169A2 (fr)

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ITMI20061772 ITMI20061772A1 (it) 2006-09-18 2006-09-18 Procedimento per la preparazione di cellule epatiche da cellule progenitrici presenti nel sangue di cordone ombelicale
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013190013A1 (fr) * 2012-06-19 2013-12-27 Waclawczyk Simon Cellules de phénotype hépatocytaire
CN109370991A (zh) * 2018-11-22 2019-02-22 上海长海医院 一种膀胱癌干细胞标志物及标记方法、膀胱癌分子分型试剂盒及应用
US11274279B2 (en) 2020-03-11 2022-03-15 Bit Bio Limited Method of generating hepatic cells

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1578956A4 (fr) * 2002-11-13 2006-02-08 Univ Washington Lignees cellulaires hautement permissives pour replication de l'arn du virus de l'hepatite c

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013190013A1 (fr) * 2012-06-19 2013-12-27 Waclawczyk Simon Cellules de phénotype hépatocytaire
WO2013189521A1 (fr) * 2012-06-19 2013-12-27 Waclawczyk Simon Procédé de génération de cellules de phénotype hépatocytaire
CN109370991A (zh) * 2018-11-22 2019-02-22 上海长海医院 一种膀胱癌干细胞标志物及标记方法、膀胱癌分子分型试剂盒及应用
US11274279B2 (en) 2020-03-11 2022-03-15 Bit Bio Limited Method of generating hepatic cells

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WO2008035169A3 (fr) 2008-06-12

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