RO126746B1 - In-vitro process for simultaneously obtaining hepatocytic and pancreatic progenitors from stem cells isolated from human placenta - Google Patents

Abstract

The invention relates to a method for preparing hepatocytic and pancreatic progenitors to be used in cellular therapy. According to the invention, the method consists in growing stem cells isolated from the chorial or membranous portion of the placenta, in usual laboratory conditions, after which there takes place the expansion of undifferentiated stem cells and characterization thereof, further on they being grown on a collagen substrate, in the presence of a differentiation medium, resulting in a change of cell morphology and three-stage differentiation of placenta mesenchymal stem cells into common hepatic-pancreatic progenitor cells.

Description

The invention relates to a process of concomitant production of liver and pancreatic cells from stem cells isolated from the human placenta, with possibilities for use in cellular treatment.

Although knowledge about biology, behavior, potential of stem cell applications in the human clinic has gained substantial momentum in recent years, the definition of the term stem cell is still controversial, being a developing concept [Parker M.A., Cotanche

YES, 77 potential use of stern cells for cochlear repair ^> 'Audiol. Neurootol. 2004; 9: 7280, Parolini O., Soncini M. Human placenta: A source of progenitor / stem cells? '' J. Reproductions. Endocrinol, 2006; 3: 117-126]. Stem cells are generically defined as undifferentiated cells, capable of self-regeneration, as well as differentiation into specific lineage cells. Self-regeneration can be maintained over many generations, increasing their number, but also giving rise to increasingly differentiated daughter cells, also called progenitor or progenitor cells. The latter are able to continue their differentiation, but are not capable of self-regeneration.

The following terms related to the proliferation and differentiation capacity of stem cells were thus delimited [Fortier L.A., Stern Cells: Classifications, Controversies and Clincal Applications. Vet. Surg .; 2005: (34): 415-423]:

- totipotent stem cells - which have the genetic potential to differentiate into any type of cell in the body, including placental cells and extra-embryonic tissues, thus having all the properties necessary to obtain a whole human fetus;

- pluripotent stem cells - which can be isolated from the internal mass of the blastocyst, are capable of forming the tissues originating in the 3 embryonic leaves (endoderm, mesoderm and ectoderm), less placental cells and extra-embryonic tissues;

- multipotent stem cells - are cells that can only give birth to a limited number of cell types or tissues, restricted to a particular embryonic stem, which can be identified in developing faces and adult organisms;

- unipotent stem cells - are cells that can give birth to a single cell type.

Stem cells can also be classified according to the stage of development of the organism in which they are obtained:

- embryonic stem cells - derived from the internal cell mass of the blastocyst, are pluripotent stem cells;

- fetal stem cells - such as cells harvested from amniotic fluid, which are pluripotent;

- adult stem cells - which are obtained postnatally from organisms, from tissues of endodermal, mesodermal and ectodermal origin.

These cells are pluripotent and do not express the markers specific to the totipotent cells, and do not form teratomas [Mahendera S.R.: Stern sense: a proposal for the classification of stern cells. Stern Cells and Development, 2004,13: 452-455].

Cell therapy involves the manipulation of living cells, complex systems and with unpredictable behavior under transplant conditions. In order to implement stem cell therapy in the human clinic, in-depth studies of stem cell biology are required. Stem cell manipulation raises many ethical and technical problems, and the translation from preclinical research to clinical research requires the fulfillment of some essential conditions:

- obtaining a sufficiently large number of cells;

- non-invasive methods of isolation and purification;

- increased expansion capacity (proliferation);

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- controlling their differentiation;

- immunological characterization;

- molecular and functional characterization;

- the reproducibility of the experiments;

- not to harm the host organism.

The current sources of stem cells are: embryonic stem cells and adult stem cells. Both sources have their advantages and disadvantages. Obtaining an increased number of embryonic stem cells is limited by the induction of the phenomenon of spontaneous differentiation, as well as by the limited possibilities of controlling a differentiation directed towards a particular cell type. Another disadvantage of human embryonic stem cell culture is the very low rate of stabilization of cell lines [Kodama M et al. (2009) Embryonic Stern Cell Transplantation Correlates With Endogenous Neurogenin 3 Expression and Pancreas Regeneration in Streptozotocin-injured Mice J. Histochem Cytochemj. in 2005, around 100 cell lines were stabilized worldwide, without sufficiently rigorous characterization [Tiina Matikainen, Jarmo Laine: Placenta-an alternative source of stern cells. Toxicology and Aplied Pharmacology Review (2005); 207: S544 – S549]. Another obstacle to the use of embryonic stem cells is the risk of inducing teratomas.

Adult stem cells do not present ethical problems because they are isolated from adult organisms. These cells are very small in number but sufficient to ensure tissue renewal, and to be mobilized to repair lesions in an organ or tissue, under these conditions the stem cells proliferate and differentiate into the damaged tissue cells. After the endogenous stem cell depletion, a process of recruiting non-hematopoietic stem cells from peripheral blood and bone marrow takes place. This whole process requires the release of cytokines and other growth factors [Tiina Matikainen, Jarmo Laine: Placenta-an alternative source of stern cells. Toxicology and Aplied Pharmacology Review (2005); 207: S544-S549],

The major disadvantage of adult stem cells is that they have a much lower differentiation potential than embryonic stem cells, as well as the fact that they are rare cells (1:10 ⁷ ... 10 ⁸ of the total cells) [Parolini O., Soncini M Humanplacenta: A source ofprogenitor / stem cells ?, J. Reproductions. Endocrinol, 2006, 3: 117-126]. Mesenchymal stem cells (CSM) are multipotent stem cells that can differentiate into a variety of cell types, in addition to hematopoietic cells: osteoblasts, chondrocytes, myocytes, adipocytes, neuronal cells; In the last decade an unexpected evolution has been observed in the study of stem cell biology, as more and more evidence is accumulating that demonstrates a much greater plasticity of adult stem cells than the criteria established by the definition imposed by embryonic stem cells [Phinney

G. D., Prokop D. J.: Concise review: Mesenchymal stem / pluripotent stromal cells: the state of transdifferentiation and modes oftissue repair-currenC Stern Cells, 2007; 25: 2896-2902], at the same time efforts are being made to decipher the molecular mechanisms that dictate the plasticity of these cells, as well as to develop exploitation modalities in cell therapy. In the human clinic, the most common source of CSM is the adult bone marrow. The percentage of CSM in bone marrow is quite low (0.001 ... 0.01%) [Pittenger M. F., Mackay A. M., Beck S. C., et al. Multilineage potential of adult human mesenchymal stern cells. Science, 1999; 284: 143-147], and it decreases with age. A disadvantage of this source is also that bone marrow harvesting is an invasive maneuver.

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An already widely used alternative is umbilical blood, but the percentage of CSM is also very low. From a total number of nucleated cells obtained from 1-2 x 10 ^{9 the} number of CD 34 + cells is 2-3 x IO ⁶ [Kodama M et al. (2009) Embryonic Stern Cell Transplantation Correlates With Endogenous Neurogenin 3 Expression and Pancreas Regeneration in Streptozotocin-injured Mice J. Histochem Cytochem]. Hematopoietic stem cells isolated from umbilical blood are used as an alternative method for bone marrow transplantation in hematological malignancies. The major disadvantage of using umbilical blood as a source of stem cells is the small number of cells that can be obtained. Much richer in CSM is the amniotic fluid, which contains a heterogeneous population of cells of fetal origin.

Placenta is one of the most advantageous sources, much studied lately, fulfilling the 2 main goals of cell therapy: obtaining as many cells as possible, and using non-invasive methods for harvesting them.

The formation of the placenta is essential for the pregnancy to evolve normally. After fertilization, implantation requires trophoblast differentiation, followed by rapid assembly of embryonic cells into a functional placenta [Kodama M et al. (2009) Embryonic Stern Cell Transplantation Correlates With Endogenous Neurogenin 3 Expression and Pancreas Regeneration in Streptozotocin-injured Mice J Histochem Cytochem]. The ectodermal cells, also called the cytotrophoblast, will anchor the placenta to the intrauterine circulation, allowing the transport of nutrients and blood gases, as well as the elimination of toxins. Placenta also plays the role of hematopoietic extramedullary organ during early embryogenesis. In addition to hematopoietic stem cells, the placenta also contains a population of multipotent stem cells, which express some of the markers of embryonic stem cells: c-kit, Oct 4, Sox 2, SSEA-1, SSEA-3, 4 and alkaline phosphatase. These cells resemble mesenchymal stem cells and in that they can be differentiated into liver, endothelial, pancreatic, neuronal cells [Strom S., Miki T., "Placenta! derived stern cells and uses thereof ', United States Patent Application Publication US 2003/0235563 A1j.

Liver disease accompanied by liver failure is an important problem in clinical pathology, and one of the treatment options with attempts at curability is organ transplantation, a difficult method involving the presence of a compatible donor. That is why alternatives to organ transplantation are sought, such as therapies based on hepatocyte transplantation or implantation of hepatocellular constructs. The primary culture of normal hepatocytes is extremely demanding, starting from the intraoperative harvesting procedure, to the importance of the microdomain signals, the extracellular matrix and soluble mediators. Thus, the construction of a liver tissue engineering platform requires in-depth knowledge and controlled reconstruction of these complex environmental factors [Culture of Cells for Tissue Engineering Gordana Vunjak-Novakovic, R. lan Freshney, 2006 WileyLiss, Chap. 15: 417-473 Tissue Engeneering of the Liverj.

The main purpose, regarding the treatment of diabetes, is to achieve an optimal level of blood sugar, avoiding hypoglycemic episodes. Experiments with beta-cell transplantation in diabetic patients have shown that this type of treatment can be a solution. As the isolated beta cells in the pancreas are in a limited quantity, new sources of stem cells are needed from which to obtain insulin-producing cells and new protocols. Promising results, but still in need of improvement, were obtained using the source of embryonic and adult stem cells.

In terms of embryonic cells, a number of differentiation protocols were used, all with variable results [Kodama M et al. (2009), Embryonic stern Cell Transplantation Correlates With Endogenous Neurogenin 3 Expression and Pancreas Regeneration in Streptozotocin-injured Mice J Histochem Cytochem, Parekh VS et al. (2009) Differentiation of human umbilical cord blood-derived mononuclear cells to endocrine pancreatic lineage., Differentiation 24: 105-227], It must be said that in this case, as in all cases using embryonic cells, they have advantages, but also a number of disadvantages. Their special plasticity makes them difficult to control, when it is desired to obtain a certain type of specialized cell, because it involves the control of all intracellular signaling pathways, which is impossible for researchers today. This explains the partial and controversial results obtained using embryonic stem cells, as well as the discrepancy between their theoretical potential and practical practical results in their use in therapy [Jenifer M et al., 2008, On the origin ofthe beta cells, Genes and Development 22: 1998-2021].

Regarding the placenta, as a source for obtaining insulin secretory cells, the number of studies is much lower. There are mainly two studies, both of which are from Chinese researchers. In the first, Sun NZ [Sun NZ et al. (2009), In vitro differentiation of human placenta-derived adherent cells into insulin-producing cells J Int Med Res 37 (2): 400-6] uses an extreme protocol. simply, at least in what he declares. The medium is classic, Dulbecco's modified Eagle medium with high glucose concentration, with 10% bovine serfetal and 1% non-essential amino acids, beta-mercaptoethanol and glutamine, to which only adds bFGF, the basic growth factor of fibroblasts. The cells are passed at 2 ... 3 days and observed under the microscope in inverted phase daily. Although the protocol is extremely simple compared to the other protocols used, the researchers claim that they obtained gene expression for Pd x 1, insulin 1 and insulin 2 at 7 and 14 days [Chang CM etal, (2007) Placenta-derivedmultipotentstern cells induced to differentiate into insulin-positive cells. Biochem Biophys Res Commun 357: 414-20], using cells isolated from the placenta, uses a more complex protocol: medium is Eagle's medium modified by Dulbecco / medium F 12 1: 1, with ITS (insulin-transferin selenium), 0.6 % glucose, 25 g / ml insulin 100 g / ml transferin, 20 nM progesterone, putrescine, 30 nM selenium chloride, 2 mM glutamine, 3 mM sodium bicarbonate, 20 ng / ml Epithelial growth factor, 20 ng / ml Factor fibroblast growth basic, 20 ng / ml Hepatocyte growth factor.

In order to have information on the state of the research and the invented patents, researches were carried out in the databases for patents, and the following two patents were considered to be close to the proposed invention: US 2010112697 (A1) and WO 03042405 (A2).

US Patent No. 2010112697 (A1) relates to a method for isolating mesenchymal stem cells derived from the chorionic plate. The method includes: harvesting the chorionic membrane plate, and isolating and culturing the cells in an environment containing a bovine serfetal and an antibiotic. This patent claims only methods for obtaining mesenchymal stem cells, in particular by mechanical and enzymatic treatments, and the description refers to methods of morphological and phenotypic characterization, without highlighting their ability to differentiate into multiple lineages.

WO 03042405 (A2) discloses a method of isolation, expansion and differentiation of fetal pluripotent stem cells from the chorionic area, amniotic fluid and placenta, and their use in cell therapy. Also, WO 03042405 (A2) claims the acquisition and expansion of a population of fetal pluripotent stem cells, through

RO 126746 Β1 the selection of C-kit positive cells from the level of chorionic villi, from amniotic fluid or placenta. Under the action of inductive differentiating agents, these cells were differentiated into cells with osteogenic, hematopoietic, adipogenic, myogenic, hepatic, neurogenic and endothelial phenotypes. The authors of the invention describe a possible application of stem cells isolated with monoclonal antibodies c-kit, and differentiation to the liver lineage. The disadvantage of the procedure presented in US 2010112697 (A1) is the use as a substrate for hepatic differentiation of the Matrigel, and the use of high doses of Fibroblast Growth Factor, 100 ng / ml, and Hepatocyte Growth Factor of 20 ng / ml, which increase the costs of the method. Also, this process does not offer the simultaneous acquisition of the hepatic and pancreatic progenitors, which is a novelty element of the proposed invention.

The technical problem solved by the proposed invention is to carry out a process of concomitant production of liver and pancreatic cells from the stem cells isolated from the human placenta, to provide the possibility of obtaining a sufficiently large number of cells, with increased proliferation capacity. , with controlled differentiation, and with good immunological, molecular and functional characterization, so that they can be used in cell therapy.

The process of concomitant in vitro production of hepatocyte and pancreatic progenitors from stem cells isolated from the chorionic or membranous portion of the human placenta consists in the fact that, in the first stage, common hepato-pancreatic progenitors are obtained by culturing the expanded cells for 16 years. .48 h, on plates with collagen and laminin substrate, in the presence of a differentiation medium H1, followed by processing of cells that adhered to the first stage collagen and laminin plates, in the presence of an H2 medium, for 3 ... 4 weeks, with the change of the H2 medium every 3 ... 4 days, resulting in hepatocyte progenitors, and finally, processing of the cell suspension resulting in the first stage, resulting in pancreatic progenitors.

Application of the invention brings the following advantages:

- The use of placenta cells isolated from the placenta is an accepted source, and eliminates the shortcomings of the other stem cell sources (non-invasive sampling, low immunogenicity, the possibility of obtaining a large number of adult stem cells, even from the isolation phase);

- the concomitant production of common progenitor cells for the hepatic and pancreatic lineage, thus allowing the same progeny of cells to produce both hepatic progenitor cells and pancreatic progenitor cells, which can then be used in cell therapies, leading to reagent economics, and of time;

- the method lasts 21 ... 28 days until completely differentiated and functional cells are obtained;

- it does not require special cultivation conditions, it can be carried out under the conditions of a classical cell culture laboratory, without the need for additional expensive equipment;

- allows the modulation of the differentiation and the desired stage according to the needs.

The following is an example of the in vitro differentiation process of placental stem cells, which produces an increased and homogeneous rate of common progenitor cells, with double differentiation potential for both hepatocytes and functional pancreatic beta cells.

The process involves several steps.

Step 1 - Isolation of stem cells from the placenta

Harvesting of the term placenta is performed in sterile conditions, from birth to term, by natural route or by caesarean section, by placing it on ice, in sterile containers. The placentas, fetal membranes and umbilical cord are washed outside with a sterile, cold, phosphate-buffered saline solution for removal of blood, and then placed in ethylene oxide sterilized plastic containers, immersed in phosphate-buffered, sterile, cold saline at 4 ° C. The processing of fetal attachments is performed within 3 to 24 hours, without a

RO 126746 Β1 major influence on the viability of the cells and the possibility of their cultivation. The amniotic membrane is detached from the underlying chorion, and the two components are then mechanically and enzymatically processed into separate containers. Epithelial cells from the amniotic membrane (MAE) are extracted by enzymatic digestion with 0.05% trypsin / EDTA-4 (Ethylenediaminetetraacetic acid) 0.53 mM Na, in 3 steps of 10, 30 and 30 min. Amniotic mesenchymal cells (MAM) are isolated by 2 steps of enzymatic treatment: 0.25% trypsin treatment for 5 minutes and 0.25% trypsin + 0.1% type IV collagenase treatment for 5 minutes. . Chorionic mesenchymal cells (MC) are obtained by mechanical processing + enzymatic digestion with dysplasia + type IV collagenase, for 30 min. The isolated cells are then seeded on 25 cm ² Cole vial culture plates in culture medium.

Stage 2 - Stem cell expansion in culture, and their characterization by highlighting the expression of pluripotency markers

Cultivation of cells isolated from the different components of the placenta is carried out at 37 ° C, in a CO ₂ atmosphere 7%, 99% humidity. on the 7th day after isolation, after highlighting the adhesion of the first cells, the culture medium is discarded, and then the environment is changed to

3 ... 4 days, until a cell confluence of 70 ... 80% is reached. Stem cell culture medium is Dulbecco's modified Eagle's medium 4.5 g glucose / l: F-12 medium ratio 1: 1, 15% fetal bovine serum, 1% L-glutamine, 1% penicillin-streptomycin, 1% non-essential acids, 55 μΜ beta-mercaptoethanol, 1 mM sodium pyruvate.

The expanded cells will pass when the subconfluence is reached, by detaching the cells with trypsin-Ethylenediaminetetraacetic acid. The cells were characterized by immunocytochemical staining and flow cytometry. The positivity of the isolated cells for the specific markers of mesenchymal stem cells is illustrated in the following table.

Specific markers of adult stem cells that were expressed by cells isolated from the human placenta

Surface markers Membrane stem cells (MAM) Corion stem cells CD 29 + + CD 105 + + SSEA-4 + + Nanog + + Sox 2 + + F Go + + CD 45 - - CD 14 + + CD 49 e + + CD 34 - - CD 73 + + Oct 3/4 + +

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Step 3 - Differentiation of placental mesenchymal stem cells to common hepato-pancreatic progenitor cells, by culturing on collagen + laminin substrate

This stage lasts 16 ... 48 h, in the presence of differentiation medium H1.

Sow 2-3 x 10 ⁵ stem cells isolated from the placenta, at passage 4, on plates with collagen + laminin substrate. The culture medium up to the confluence is the usual culture medium of undifferentiated stem cells: Eagle's medium modified by Dulbecco 4.5 g glucose / l: F-12 ratio 1: 1, 15% bovine serfetal, 1% L-glutamine, 1 % + penicillin-streptomycin, 1% non-essential acids, 55 μΜ beta-mercaptoethanol, 1 mM sodium pyruvate.

when the confluence is reached, change of medium with medium H1: Eagle's medium modified by Dulbecco 1g glucose / 1: MCDB201 ratio 1: 1.1%, 4.7 pg / ml linoleic acid, 1% insulin-transferin-selenium, Factor growth factor from blood platelets 10 ng / ml, Epithelial growth factor 20 ng / ml, 10 nM dexamethasone, 10 mM ascorbic acid, 1% L-glutamine, 1% penicillin-streptomycin, 1% non-essential amino acids.

Preparation of collagen substrates with laminin

Collagen - a type IV collagen solution is prepared, at a final concentration of 15 pg / ml. Petri dishes and plates with a diameter of 6 cm were used. The final solution for collagen is placed in 6-well plates, each 1 ml / well, and left for 1 h in the laminar flow niche. The excess solution is discarded, leaving only a thin blade of liquid on the bottom of the plate. The plates are allowed to dry and then sterilized with ethylene oxide.

Laminin - the stock solution (see explanation in the claims) of 1 mg / ml stored at -20 ° C is thawed at 2 ... 8 ° C. The dilution is carried out in phosphate-buffered saline, having brought to a solution of 100 µg / ml. Calculate the required amount of solution for each surface so that at the end the concentration is 2 pg / cm ² . The pre-sterilized collagen-coated plates are incubated with the laminin solution for 2 hours at 37 ° C, then washed 2X with phosphate-buffered saline, and prepared for cell seeding.

The differentiation to the hepatic and pancreatic progenitors is presented in relation to fig. 1 ... 4, which represents:

FIG. 1, gene expression at different stages and conditions, during hepatic differentiation, as compared to the hepatocarcinoma cell line HepG2 (F1BH - stage 1, 16 h, 100 ng Epidermal growth factor; F1BL - shortening we used to identify - cells subjected to the hepatic differentiation protocol (F) in the first stage (1), with a short duration of action of growth factors (BL) - stage 1, 16 h 20 ng Epidermal growth factor; F11B - cells subjected to the hepatic differentiation (F) in the first stage (1), with a long duration of action of growth factors (B) - step 2, 72 h; F2B cells subjected to the hepatic differentiation protocol (F) in the second stage (2), with a long duration of action of growth factors (B) - stage 2, with hepatocyte growth factor and oncostatin M; F2BR - cells subjected to the hepatic differentiation protocol (F) in second stage (2), with a long duration of action of growth factors (B) and rifampicin (R) -stage 2 with hepatocitaroncostatin growth factor M and rifampicin);

FIG. 2, cells isolated from differentiated chorion in H2 medium, 30 days for cultivation; coloring for cytokeratin 18 marked with FITC;

FIG. 3, results obtained, highlighted by dithizonone staining;

FIG. 4, the diagram of the hepato-pancreatic double differentiation.

Stage 4 - Differentiation to hepatic progenitors

After exposure to the H1 medium, there is a change in the morphology of the cells with the rounding of the shape, as well as the detachment of cell groups that form spherical aggregates. After 16 ... 48 h the medium is changed, with the taking of the cells in suspension, which will be transferred to the plates with collagen + laminin, to induce pancreatic differentiation. The adhered cells, remaining on the plates with collagen + laminin, will undergo the second stage of differentiation to hepatocytes, in the presence of medium H2, medium identical to H1, to which it is added, as factors of

RO 126746 Β1 additional growth: 4 ng / ml Hepatocyte growth factor and 4 ng / ml Oncostatin M, 1% Lglutamine, 1% penicillin-streptomycin, 1% non-essential amino acids. This stage lasts 3 ... 4 weeks, with the change of H2 environment every 3 ... 4 days. To the H2 environment, there will be no addition of growth factor from blood platelets, and epithelial growth factor. These cells adhered to at the end of the 3 to 4 week period will express hepatocyte differentiation markers.

Following the experiments performed, it was observed that a concentration of epithelial growth factor 20 ng / ml is sufficient to induce differentiation to hepatic progenitors in the first stage, as shown in fig. 1 (genetic analysis by the polymerase chain reaction method, preceded by the use of reverse transcriptase, of cells adhered to the plaque, compared to the hepatocarcinoma cell line HepG2). From the same analysis, it was concluded that an incubation time of 16 h is sufficient to induce differentiation (F1BL sample), as these cells begin to express the genes for cytokeratin 18. From the same figure it is concluded that cells grown in the second stage of differentiation express more intense pancreatic lipase and fetoprotein, in the case of the environment protocol containing hepatocyte growth factor and Oncostatin M. (F2BR sample).

The presence of cytokeratin 18 was also confirmed by immunocytochemical staining (Fig. 2).

Stage 5 - Differentiation towards pancreatic progenitors

This stage is carried out in 3 phases:

a) The cell suspension taken from the collagen + laminin plates is centrifuged for 4 min at 500 rpm and the cell sediment is resuspended in P1 medium, and sown on the collagen + laminin plates. The cultivation period is 4 ... 7 days. The cells remain suspended, but their morphology changes, the cell aggregates become denser, more condensed, with a larger spherical shape.

Eagle medium modified by Dulbecco / MCDB201 (final glucose content 6.5 g / l), 1% bovine serum albumin, 1% Supplement N1.0.5% B27.10 ng / ml Fibroblast growth factor, 10 ng / ml Basic fibroblast growth factor, 4 ng / ml Hepatocyte growth factor, heparin, 1% insulin-transferin-selenium, 1% L-glutamine, 1% penicillin-streptomycin, 1% non-essential acids.

b) The cell suspension thus cultured is taken up and centrifuged, resuspended in P2 medium, and sown on other plates with collagen + laminin. Cultivation in P2 environment is

6 ... 7 days. Medium P2 is composed of low concentration glucose solution Eagle's medium modified by Dulbecco / MCDB 201 (1 g / l glucose concentration), 10% FCS, 10 mM nicotinamide, 1% Glutamine, 1% penicillin-streptomycin, 1% non-essential acids.

c) The cell suspension thus cultured is taken up and centrifuged, resuspended in P3 medium, and sown on other plates with collagen + laminin. Cultivation in the P3 environment is

6 ... 7 days. Environment P3 is composed of Dulbecco / MCDB201 modified Eagle's Environment (glucose 1 g / l), 10% bovine serfetal, 10 mM eExendin 4, 1% Glutamine, 1% penicillin-streptomycin, 1% non-essential amino acids. At the end of 6 to 7 days, glucagon and transforming growth factor beta are added to the medium.

Staining with dithisone - a dye used for highlighting beta islands, at the end of the P3 medium cultivation period of cell aggregates derived from stem cell populations, has been shown to obtain positive cells for dithisone staining, both in number, as well as as a percentage of positivity (fig. 3). in FIG. 3, we analyzed the morphological aspect of the stem cells isolated from the chorion and the amniotic membrane, exposed to a sequential differentiation: medium H - medium P1 - medium P2 - medium P3. The presence of spherical cell aggregates in suspension is observed, some of them being positive for dithizonis (red color).

Fig. 4 briefly illustrates the sequence of administration and the composition of the media intended for each stage of differentiation.

Claims (7)

claims 1. Process for concomitant in vitro production of hepatocyte and pancreatic progenitors from stem cells isolated from the chorionic or membranous portion of the human placenta, consisting of stages of isolation, expansion and differentiation of cells isolated in different culture media, characterized in that in the former stage is obtained common hepato-pancreatic progenitors, by cultivating expanded cells, for 16 ... 48 h, on plates with collagen and laminin substrate, in the presence of a differentiation medium H1, followed by processing of cells that adhered to the plates with collagen and laminin from the first stage, in the presence of a medium H2, for 3 ... 4 weeks, with the change of the medium H2 every 3 ... 4 days, resulting in hepatocyte progenitors, and finally the processing of the cell suspension in the first stage , resulting in pancreatic progenitors. 2. Process according to claim 1, characterized in that the processing of the cell suspension, for obtaining pancreatic progenitors, consists of: a) centrifugation of the cell suspension 4 min at 500 rpm, resuspending the suspension in a P1 medium and cultivation 4 ... 7 days on the collagen and laminin plates; b) resuspending the cell suspension resulting from phase a), in a P2 medium, and culturing 6 ... 7 days on collagen and laminin plates, resuspending the cell suspension resulting from phase b), in a P3 medium, and cultivating 6 ... 7 days on collagen and laminin plates. Process according to Claim 1, characterized in that the H1 medium consists of Dulbecco's modified Eagle's medium 1 g glucose / l: MCDB201 medium in a ratio of 1: 1, 1% bovine serum albumin, 4.7 µg / ml acid linoleic, 1% insulin-transferinelene, 10 ng / ml Blood platelet growth factor, 20 ng / ml Epithelial growth factor, 10 nM dexamethasone, 10 mM ascorbic acid, 1% L-glutamine, 1% penicillin-streptomycin, 1% non-essential acids. 4. Process according to claim 1, characterized in that the H2 medium consists of 4 ng / ml Hepatocyte growth factor and 4 ng / ml oncostatin M, 1% L-glutamine, 1% penicillin-streptomycin, 1% non-essential amino acids, Eagle's medium modified by Dulbecco 1 g glucose / l: medium MCDB201 ratio 1: 1, 1% bovine serum albumin, 4.7 pg / ml linoleic acid, 1% insulin-transferin-selenium, 10 ng / ml growth factor derived from blood platelets, 20 ng / ml Epithelial growth factor, 10 nM dexamethasone, 10 mM ascorbic acid, 1% L-glutamine, 1% penicillin-streptomycin, 1% non-essential acids. 5. Process according to claim 2, characterized in that the medium P1 consists of the Dulbecco modified Eagle medium with high glucose: the medium MCDB201 the final glucose amount being 6.5 g / l, 1% bovine serum albumin, 1% supplement N1, 0.5% B27, 10 ng / ml epidermal growth factor, 10 ng / ml fibroblast growth factor, 4 ng / ml hepatocyte growth factor, heparin, 1% insulin-transferin-selenium, 1% L -glutamine, 1% penicillin-streptomycin, 1% non-essential amino acids. 6. Process according to claim 2, characterized in that the medium P2 consists of the Dubelcco modified Eagle medium with low glucose: MCDB 201 with glucose of 1g / l, 10% fetal bovine serum, 10 mM nicotinamide, 1% L- glutamine, 1% penicillin, streptomycin, 1% non-essential amino acids. 7. Process according to claim 2, characterized in that the P3 medium consists of: Dubelcco's modified low glucose Eagle medium: MCDB201 medium with glucose 1 g / l, 10% fetal bovine serum, 10 mM exendin, 1% L -glutamine, 1% penicillin-streptomycin, 1% non-essential amino acids.