KR101873430B1 - Methods for Differentiating Stem Cells To Hepatocytes Using Biocompatible Solubilized Scaffold Concentrate Derived from Decellularized Organ Tissue - Google Patents
Methods for Differentiating Stem Cells To Hepatocytes Using Biocompatible Solubilized Scaffold Concentrate Derived from Decellularized Organ Tissue Download PDFInfo
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Abstract
The present invention relates to a method for differentiating stem cells into hepatocytes using a biocompatible solubilized scaffold extract derived from a defatted living tissue, and a composition for inducing differentiation. The method for differentiating stem cells into hepatocytes using the defatted biomedical tissue-derived biocompatible solubilizing scaffold extract and the medium composition for inducing the differentiation of stem cells into hepatocytes is a method for producing a degenerated liver slice Can provide biomimetic 2D and 3D microenvironment for the survival and differentiation of stem cells into stem cells and can be effectively used for the differentiation and culturing of degenerated stem cells.
Description
The present invention relates to a method for differentiating stem cells into hepatocytes using a biocompatible solubilized scaffold concentrate derived from a defatted living tissue.
Orthotopic liver transplantation is the only standard treatment for patients with end-stage liver disease. There are serious problems with these procedures, such as the need for donor liver, the lack of liver donors, as well as post-transplant rejection. One potential way to complement the chronic shortage of donor agencies is to develop between bioartificial liver and secondary livers. The liver is very complex in structure and has various functions within the body, including the production of biochemicals necessary for detoxification, protein synthesis and degradation. Therefore, it is difficult to manufacture a scaffold that is the same as the liver in terms of components, structure and function. It also has good biocompatibility, suitable porosity, and should be similar in composition, morphology and structure to the living body. Thus, the functional biomechanical liver fabrication for tissue engineering, organ transplantation and drug screening can be constructed using a de-saturation process. When the intracellular components are completely removed from the liver tissue using a detergent, the structural and functional characteristics of the intact liver-specific ECM and vascular network are well preserved. The application potential of de-saturated matrix in tissue engineering has been identified for a number of tissues including bladder, artery, esophagus, skin and organs. Degeneration of tissues or organs has been studied as a promising approach to the production of functional scaffolds for cell culture and transplantation.
In order to develop artificial liver for regenerative medicine and tissue engineering, two basic and complementary key elements are required. The first is a biologically compatible scaffold that conserves all extracellular matrix proteins and mimics natural liver structures. A number of synthetic or naturally derived substrates have been used in liver tissue engineering. However, most synthetic substrates were biocompatible. The depleted hepatic scaffold is the most suitable and reliable source for maintaining extracellular matrix, proteins and blood vessels. The second is a suitable cell source for reseeding that can support normal liver functioning. Cells constituting the tissue are essential for successful tissue regeneration. Considering proliferative activity and cell differentiation potential, stem cells are actually promising. Self-renewal is a characteristic of stem cells that imparts multi-potential differentiation ability to stem cells. There are many cell sources such as adult stem cells, fetal hepatic progenitor, and pluripotent stem cells that can differentiate into hepatocytes. However, the possibility of adult stem cell differentiation into hepatocytes is still controversial, and pluripotent stem cells are promising for hepatocyte differentiation. On the other hand, hepatocytes separated and cultured in a plastic in vitro from liver tissue are fragile. These cells rapidly lose their liver-specific function and, due to lack of proper environmental basis, tend to die of apoptosis.
Induced pluripotent stem cells (iPS) have the ability to proliferate indefinitely on in vitro while retaining the potential to differentiate into hepatocytes on in vitro and in vivo, The best model for treatment is considered.
However, degenerative stem cells are currently the subject of customized stem cells because they have no immune rejection response and are capable of infinite supply. However, they have not been put to practical use due to various limitations.
One problem is that the efficiency of differentiation into specific cells is low. In liver cells differentiated from degenerated stem cells, the efficiency of liver-specific marker expression or function is very low as compared with the actual hepatocyte. Therefore, it is necessary to develop a technique for increasing the differentiation efficiency for practical use. It is insufficient.
Recent studies have focused primarily on the application of depleted whole liver structures. In addition, the versatility of de-saturated liver tissue for the production of coating materials or other types of biomaterials such as injectable hydrogels is being studied. Obtaining and using natural ECMs from specific tissues can provide an ideal growth environment as they can mimic the cell-specific in vivo microenvironment more closely. With the development of de-saturation techniques, tissue-specific extracellular matrix (ECM) as a whole new biomaterial has attracted the attention of many researchers. Although collagen, fibronectin, bitonectin, laminin or matrigel have been used as coating materials in a variety of combinations of single proteins, they have been shown to encapsulate in vivo ECM completely in terms of tissue-specific binding or the ratio of various proteins and polysaccharides Do not. ECM scaffolds and substrates are ideal candidates for tissue engineering. ECM constitutes a network of occlusion of fibrous proteins such as collagen, elastin, fibronectin, laminin and glycosaminoglycan (GAG). In addition, ECM directs important morphological and physiological functions by binding of growth factor (GF) and interaction with cell-surface receptors to induce signal transduction and regulate gene transcription.
The present inventors developed an elaborate platform from the extracellular matrix to provide the above two functions and provide a biocompatible two-dimensional matrix coating material and a three-dimensional biomatrix for degenerative stem cell culture . In addition, iPS cells were inoculated into the 3D biomatrix to induce differentiation into hepatocytes, and the biocompatibility and survival rate of induced hepatocytes were confirmed. Thus, de-saturated liver slices can provide efficient and biomimetic 2D and 3D microenvironment for iPS survival and hepatocyte differentiation, indicating applicability in liver tissue engineering.
The present inventors have made efforts to develop a method for culturing degenerated stem cell (iPS) or differentiation into hepatocytes using a new biocompatible material derived from a living tissue. As a result, an elaborate platform has been developed from the extracellular extracellular matrix to produce a biocompatible two-dimensional matrix coating material, a liver extracellular matrix (LECM); Hereinafter, a biocompatible solubilized scaffold concentrate derived from degenerated biotissue) and a 3-dimensional biomatrix are provided, and iPS cells are differentiated into hepatocytes by using the same, and the differentiated hepatocytes (iPSC- Heps) and biocompatibility and transplant survival rate.
Accordingly, an object of the present invention is to provide a method of differentiating stem cells into hepatocytes.
Another object of the present invention is to provide a medium composition for inducing the differentiation of stem cells into hepatocytes.
Hereinafter, the present invention will be described in detail.
According to one aspect of the present invention, the present invention provides a method of differentiating stem cells into hepatocytes comprising the steps of:
(a) forming and culturing embryoid bodies (EBs) by culturing stem cells in a culture container coated with a coating substrate and / or supporting cells;
(b) inducing differentiation into hepatocytes by adding the differentiation inducing substances in the step (a) stepwise; And
(c) adding a biocompatible solubilized scaffold concentrate derived from the defatted biomaterial to induce the differentiation of step (b), thereby enhancing the differentiation rate of hepatocytes.
The culture container of step (a) is coated with a coating substrate and / or supporting cells. The coating substrate and supporting cells may be any material or support cell known in the art for culturing and differentiating cells.
In the present invention, the culture container is coated with at least one selected from the group consisting of gelatin, matrigel, collagen and STO feeder cells, and preferably the culture container is coated with matrigel and collagen.
According to a preferred embodiment of the present invention, the stem cells of step (a) may be selected from the group consisting of Induced Pluripotent Stem Cells (iPSC), Embryonic Stem Cells, Marrow-derived Stem Cells Derived stem cells, placenta-derived stem cells, adipose tissue-derived stem cells, placenta-derived stem cells, and the like, and may be appropriately selected depending on the purpose have.
In the present invention, the stem cells are preferably Induced Pluripotent Stem Cells (iPSC), Embryonic Stem Cells or Marrow-derived Stem Cells, and most preferably, Induced Pluripotent Stem Cells (iPSC).
According to a preferred embodiment of the present invention, the step (b) of inducing differentiation into hepatocytes comprises the steps of: (a) adding Y27362 and / or activin A;
(B) adding bone morphogenetic protein 4 (BMP4) and / or basic fibroblast growth factor (bFGF);
(C) adding HGF (porcine hepatocyte growth factor) and / or ITS (insulin-transferrin-selenium X); And
(D) adding at least one selected from the group consisting of OSM (oncostatin M), DEX (dexamethasone) and ITS (insulin-transferrin-selenium X);
, And the steps may be repeated in sequence or simultaneously.
More preferably, the step (b) of inducing differentiation into hepatocytes comprises the steps of: (1) culturing a hepatocyte selected from the group consisting of oncostatin M, dexamethasone, and insulin-transferrin-selenium X And most preferably OSM (oncostatin M), DEX (dexamethasone) and ITS (insulin-transferrin-selenium X).
In one embodiment of the present invention, the differentiation inducing step is used as a four-step differentiation protocol (FIG. 1D). As the first step, 100 ng / ml Actin A (Act A) is treated with 10 mM Y27362 on the first day, and on the 2-5th day, differentiation medium with 100 ng / ml Actibin A is used. As a second step, 20 ng / ml BMP4 (bone morphogenetic protein 4) and 10 ng / ml bFGF are then fed for 5 days. As a third step, 20 ng / ml porcine hepatocyte growth factor (HGF) and insulin-transferrin-selenium X (Gibco / Life Technologies) are used at 10-15 days. As a fourth step, cells are cultured for 5 days with 20 ng / ml OSM (oncostatin M) (ProSpec), 10-7 M DEX (dexamethasone) (Sigma-Aldrich), and ITS.
According to a preferred embodiment of the present invention, the solubilized scaffold concentrate of step (c) is incorporated at 1% to 40% of the total volume of the medium, more preferably 1% to 20% 5% to 15%, and most preferably 10%.
In the present invention, the solubilized scaffold concentrate of step (c) is prepared by the following steps:
(c-1) decellularizing the biotissue;
(c-2) obtaining an extracellular matrix (ECM) from the degummed tissue;
(c-3) lyophilizing the ECM of the step (c-2) and pulverizing the powder to form a powder;
(c-4) obtaining an acid-soluble extract obtained by adding an acid solution to the powder of the step (c-3) to obtain an acid-extracted extract;
(c-5) obtaining pepsin solubilized extract by the addition of pepsin to the acid-soluble extract of step (c-4);
(c-6) neutralizing the pepsin-solubilized extract of step (c-5) to obtain a soluble extract;
(c-7) concentrating the soluble extract of step (c-6) to obtain a biocompatible solubilized scaffold concentrate.
The biotissue of the step (c-1) of the present invention is a tissue isolated from a mammal, and may include tissues of any mammal so long as it can be depolarized to obtain an extracellular matrix (ECM).
The term "Biocompatible Solubilized Scaffold Concentrate " used herein to refer to the differentiation into hepatocytes in the present invention means a concentrate obtained by concentrating a biocompatible material derived from a defatted living tissue. In the present specification, LECM (liver extracellular matrix).
It is preferably a tissue isolated from pigs, cows, horses, rabbits, dogs, cats, sheep, goats, humans, non-human primates, guinea pigs or rodents, more preferably pigs or cows, to be.
According to a preferred embodiment of the present invention, the living tissue of the step (c-1) is a liver, a heart, a kidney, a stomach, a small intestine, a large intestine, a spleen, a bladder, a lung or a skin, And most preferably liver.
As used herein, the term "scaffold " refers to a structure used for tissue engineering, which uses a combination of cells and various materials by placing a living cell. The term " scaffold " Refers to a structure (template) which is formed by decellularizing a structure, that is, a biological organ to remove cells and leaving only the outline of microstructure and organs.
Thus, the term " Solubilized Scaffold Concentrate "as used herein refers to a soluble concentrate of the scaffold that is the biological construct described above.
In the present invention, the concentrate preferably concentrates the soluble extract prepared as described above twice to 100 times, more preferably 5 times to 50 times, most preferably 10 times.
According to the present invention, the solubilized scaffold concentrate of the present invention can be obtained by 1) depletion of cell constituents resulting in minimization of immune rejection upon transplantation, 2) preservation of extracellular matrix and protein involved in cell growth and differentiation, 3) Liver lobular structure is preserved, and oxygen and nutrient supply after cell implantation is possible, and original shape and structure are maintained.
According to a preferred embodiment of the present invention, the de-saturation of the step (c-1) is performed by dissolving at least one selected from the group consisting of a lysis solution, a hypotonic solution, a surfactant, RNase A and DNase I Can be used.
According to a preferred embodiment of the present invention, the surfactant may be a conventional surfactant, and its concentration is preferably 0.1 to 10%.
Preferably, the surfactant is at least one selected from the group consisting of SDS (sodium dodecyl sulfate), Triton-X, DNase and CHAPS (3- (3-cholamidopropyl) dimethylammonio) -1-propane sulfonate SDS (sodium dodecyl sulfate) or Triton-X, and most preferably SDS (sodium dodecyl sulfate).
In the present invention, the acid solution in step (c-4) is preferably at least one selected from the group consisting of hydrochloric acid, sulfuric acid and phosphoric acid.
In this step, an acid solution may be added to the scaffold at a weight ratio of 1: 1 to 100 to obtain an acid-soluble scaffold extract.
Then, pepsin is added to the acid-soluble extract at a weight ratio of 1: 1 to 100 to obtain a pepsin solubilized extract.
According to another aspect of the present invention, there is provided a method for producing a biocompatible solubilized scaffold concentrate (hereinafter, referred to as " biocompatible solubilized scaffold concentrate ") from a stem cell to hepatocyte A culture medium for inducing differentiation is provided.
As used herein, the term "media" means a medium that enables stem cell growth, differentiation and survival in vitro to be supported and includes conventional media used in the art suitable for culturing stem cells All included. Depending on the type of cells, medium and culture conditions can be selected. The medium used for the culture is preferably a cell culture minimum medium (CCMM), which generally contains a carbon source, a nitrogen source and a trace element component. For example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, aMEM (Glasgow's Minimal Essential Medium), Iscove's Modified Dulbecco's Medium, and the like.
In addition, the medium may include antibiotics such as penicillin, streptomycin, and gentamicin.
The biodegradable biocompatible solubilized scaffold concentrate of the present invention of the present invention can be used alone or in combination with other substances to form a microenvironment for culturing and differentiation of cells.
The composition of the present invention may additionally include a differentiation inducing substance as the other substance, and the differentiation inducing substance may be any of the differentiation inducing substances known in the art, and it may be added to the type and stage of the cell And may be added singly or in combination.
For example, the composition of the present invention may be used as a drug delivery system for allowing a growth factor to be transplanted into a living body by additionally attaching a growth factor necessary for cell proliferation. As a growth factor adhering to the extracellular matrix membrane, Transforming Growth Factor-a (TGF-a), Transforming Growth Factor (TGF-b), Bone Growth Factor (aFGF) (BMP), platelet derived growth factor (PDGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), hematopoietic growth factor (EPO) GM-CSF, G-CSF, NGF, and heparin-binding EGF.
That is, the composition of the present invention is a composition for culturing or differentiating stem cells, which comprises a biocompatible solubilized scaffold concentrate derived from a defatted body tissue as an active ingredient, thereby increasing the survival and differentiation ability of cells to be cultured or differentiated .
Since the composition of the present invention comprises a solubilized scaffold concentrate prepared by the method of the present invention, its redundancy is omitted in order to avoid undue complexity of the present disclosure.
The method for differentiating stem cells into hepatocytes using the defatted biomedical tissue-derived biocompatible solubilizing scaffold extract and the medium composition for inducing the differentiation of stem cells into hepatocytes is a method for producing a degenerated liver slice Can provide biomimetic 2D and 3D microenvironment for the survival and differentiation of stem cells into stem cells and can be effectively used for the differentiation and culturing of degenerated stem cells.
Figure 1 shows the production and functional properties of porcine iPSC-Hep for meta- bolization.
Figure 2 shows the liver maturation efficiency of porcine iPSC-Hep enhanced by porcine LECM.
Figure 3 shows the differentiation rate between cell culture dish processing methods.
Figure 4 shows the most effective conditions for liver differentiation by adding various concentrations of LECM to the cell culture medium.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.
Experimental Methods and Materials
All experiments were approved by IACUC (Institutional Animal Care and Use Committee) of Kangwon National University (Chuncheon, Korea).
Production and culture of pig iPSC
The inventors have produced porcine iPSCs with minor modifications to previously reported methods. Briefly, infected pigs ear fibroblast cells to retroviruses encoding human OCT4, SOX2, KLF4 and cMYC and was inoculated into 60-mm plates containing 5 days, 5X10 4 infected cells, mouse embryonic fibroblast feeder layer . The medium for iPSC formation was supplemented with 20% knockout serum replacement, 0.1 mM nonessential amino acids (all Gibco / Life Technologies, Carlsbad, CA, USA), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, USA), 10 ng / ml human recombinant bFGF (Millipore, Billerica, MA, USA) and 1% penicillin / streptomycin (Hyclone, South Logan, UT, USA) Dulbecco ' s Modified Eagle Medium). After approximately 12 days of infection, three different porcine iPSC monocolonies (FS, E3, E4, pig iPSC strain) were generated, 5% CO 2 on mouse embryonic fibroblast cell layer,
To form embryoid bodies (EBs), iPSC colonies were transferred to an ultra low adhesive dish for 5 days. To induce spontaneous differentiation, EBs were replated on cell culture plates and cultured in medium without cytokine for 15 days.
Protocol for the Differentiation of Hepatocytes from Pig iPSCs
Colonies of porcine iPSC were isolated and seeded in 60 (60 ug / ml) coated with Matrigel (Growth factor-reduced, 1:40; Corning, Tewksbury, Mass., USA) and collagen -mm plate. Roswell Park Memorial Institute 1640 (Welgene Biotech, Daegu, South Korea) containing 10% FBS (Atlas Biologicals, Fort Collins, CO, USA) was used as the basic differentiation medium.
The differentiation induction process is shown in Figure 1D. On the first day, 100 ng / ml actin A (Act A) (ProSpec, East Brunswick, NJ, USA) was used with the 10 mM Y27362 (Enzo Life Sciences, Farmingdale, . On the 2nd to 5th days, 20 ng / ml BMP4 (bone morphogenetic protein 4) (ProSpec) and 10 ng / ml bFGF were used as the second stage and then 5 days after the differentiation medium containing 100 ng / Lt; / RTI > As a third step, 20 ng / ml porcine hepatocyte growth factor (ProSpec) and ITS (insulin-transferrin-selenium X) (Gibco / Life Technologies) were used at 10-15 days. As a fourth step, cells were incubated for 5 days with 20 ng / ml OSM (oncostatin M) (ProSpec), 10 -7 M DEX (dexamethasone) (Sigma-Aldrich), and ITS.
Immunocytochemical staining
For the alkaline phosphatase (AP) staining of porcine iPSCs, nitro blue tetrazolium chloride and 5-bromo-chloro-3-indophosphoric acid toluidine salt solution (Roche, Basel, Switzerland) were used according to the manufacturer's recommendations.
For immunocytochemical staining of porcine iPSCs, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 minutes and permeabilized with 0.25% Triton X-100 (Sigma-Aldrich).
1: 200) and stage-specific embryonic antigen (SC-33759; 1: 127 100) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), SOX2 (AB5603; Primary antibodies to 4 (MAB4301; 1: 150) (all Millipore) were used to detect undifferentiated porcine iPSC.
For detection of spontaneously differentiated iPSCs, the LSAB + system HRP (horseradish peroxidase) kit (K0679; Dako, Glostrup, Denmark) was used according to the manufacturer's recommendations. (MAB1615; Millipore) for confirmation of ectoderm, a-smooth muscle actin (ab5694; Abcam, Cambridge, MA, USA) for confirmation of mesoderm and keratin7 / 17 , Temecula, CA, USA) was diluted 1: 300 with the primary antibodies.
Primary antibodies against a-fetoprotein (AFP, A8452; 1: 200) (Sigma-Aldrich) and albumin (ALB, ab112991; 1: 200) (Abcam) were incubated overnight at 4 ° C for immunocytochemical staining of porcine iPSC- ≪ / RTI > Samples were incubated with FITC (fluorescein isothiocyante) -binding secondary antibody for 30 minutes at room temperature.
Reverse transcription (RT) -PCR
RNA was extracted from the cells using Nucleospin RNA kit (Macherey-Nagel, Duren, Germany) and cDNA was synthesized using TOPscript RT dry mix (Enzynomics, Daejeon, South Korea). Ampone Taq polymerase (GeneAll Biotechnology, Seoul, South Korea) was used for PCR. Primer sequences are shown in Table 1 below.
Functional characteristics of porcine iPSC-Heps
Periodic acid and Schiff's staining (Sigma-Aldrich) were performed to detect glycogen. Cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) and incubated for 5 minutes with periodic acid and Schiff's solution for 15 minutes according to the manufacturer's recommendations. For analysis of low density lipoprotein (LDL) uptake, differentiated hepatocytes were incubated with DiI-conjugated LDL (20 μg / ml; Biomedical Technologies, Inc., Stoughton, MA, USA) for 60 min. To assess lipid storage, cells were fixed with 10% formalin for 60 min at 4 ° C and incubated in a dark room for 30 min at room temperature with 0.5% Oil Red O solution (Sigma-Aldrich). All dye solutions were freshly prepared. For analysis of indocyanine green (ICG) uptake, 1 mg / ml ICG was added to the cell culture medium for 60 min at 37 < 0 > C. To measure the cytochrome P450 (CYP) enzyme activity of iPSC-Heps, 10-50 μM of the CYP inducer rifampicin (Sigma) was added to the cell culture medium for 48 hours.
Fluorescence-activated cell sorting (FACS)
The effect of porcine LECM on liver differentiation of porcine iPSCs was analyzed on a FACS caliber system with CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). Pig iPSC-Heps were obtained by incubation with a TypLE Express (Gibco / Life Technologies) for 15 minutes at 37 < 0 > C. Single cells were collected by filtration through a 40-μm strainer (Becton Dickinson), fixed and permeabilized with Cytofix / Cytoperm solution and washed with Perm / Wash buffer (all Becton Dickinson) according to the manufacturer's recommendations. Cells were incubated with primary antibodies against AFP (1: 200) and ALB (1: 150) in 3% FBS in PBS for 20 min at room temperature and then incubated with FITC-conjugated secondary antibodies for 30 min at 4 ° C.
Western blotting
To compare ALB expression of porcine iPSC-Heps cultured with or without LECM, cells were lysed with radioimmunoprecipitation assay dissolution buffer containing protease inhibitor cocktail (Intron Biotechnology, Seongnam, South Korea) And subjected to Western blot analysis according to known methods. The polyvinylidene difluoride membrane was incubated with goat anti-ALB antibody (1: 2000) and HRP-conjugated anti-goto IgG (1: 5000; Santa Cruz Biotechnology). Protein bands were visualized by chemiluminescence (BioNote, Hwaseong, Korea). Anti-actin antibody (1: 5000; Santa Cruz Biotechnology) was used as a loading control on the stripped membrane.
Production process of pig LECM (liver ECM)
In order to investigate the effect of the biocompatible solubilized scaffold concentrate (referred to as LECM in the present invention) derived from the de-saturatedized biotissue of the present invention in hepatic differentiation of porcine iPSCs, The pig liver scaffold was made as follows:
(1) The acquisition of liver and the depletion of liver slice
All procedures were approved by IACUC (Institutional Animal Care and Use Committee) at Kangwon National University. The pigs weighing 40 kg to 50 kg were slaughtered directly, the intestines were removed, and the liver was obtained. Degasification was performed according to a known protocol. Briefly, the obtained mesenchyme was separated and trimmed with several thin sections with an average weight of 0.8 gm per piece and a size of 1x1x0.5 cm. The sections were incubated for 1 hour with 1X PBS (phosphate buffered solution) containing 500 IU / l heparin (Chungwae Pharma Co., Seoul, Korea) and 1% penicillin-streptomycin (P / S) Lt; RTI ID = 0.0 > 4 C < / RTI > The sections were also defatted with 0.1% SDS (sodium dodecyl sulfate, Sigma-Aldrich, St. Louis, Mo., USA) at 4 ° C at 120 rpm for 72 hours using a shaker. The de-saturated solution was changed every 8 hours. Finally, all sections were rinsed three times for 2 hours with PBS containing 1% P / S each to clean residual SDS in the ECM.
(2) Preparation of Solubilized LECM
The de-saturated pork liver scaffold prepared as described above was lyophilized. 1 mg of lyophilized pig liver scaffold powder was mixed with 1 mg of a solution consisting of 3 mg pepsin / 0.1 M HCl (all Sigma) and incubated at room temperature for 72 hours with shaking at 120 rpm. After complete digestion, the pH was adjusted to 7.3 by adding 1/10 of the total volume of 1 M NaCl and 10X PBS solution. The scaffold solution was concentrated using an Amicon Ultra 153K centrifugal filter (Millipore) according to the manufacturer's recommendations.
Growth factors analysis of LECM
To assess the amount of growth factors remaining in native pig liver, de-saturated liver scaffold, sterile liver scaffold, and solubilized LECM, the human growth factor array G1 (RayBiotech, Inc., Norcross, GA, USA).
Statistical analysis
Experimental data were analyzed by analysis of variance using statistical analysis system software (SAS Institute, Inc., Cary, NC, USA). P <0.05 was considered statistically significant.
Example 1. Optimization of protocols for the production of porcine iPSC-Hep and their functional characterization
In order to produce a functionally satisfactory porcine iPSC-Hep as a cell resource for recellularization, the present inventors analyzed the functional characteristics of iPSC-Hep by optimizing the protocol. Porcine iPSC colonies had a circular planar shape and expressed AP, SOX2, OCT4, Nanog, and SSEA-4 (Figure 1A). Transcript expression of SOX2, OCT4, KLF4, Nanog, REX1 and teratocarcinoma-derived growth factor (TDGF) was confirmed by RT-PCR (Fig. 1B). The developmental potential of iPSCs to differentiate into EBs on Invitro and to form these three embryonic layers is tested for immunity using neurofilament, a-smooth muscle actin and antibodies against
Example 2 Confirmation of Improvement of Maturation of Porcine iPSC-Hep by Defatted Porcine LECM Treated with Pig-Derived Growth Factor
The role of LECM in the liver differentiation of iPSCs was assessed for cells to be used for the metastasis of liver scaffolds. To determine whether the depleted saturation scaffold affects liver differentiation of porcine iPSCs, LECM was prepared from adult porcine liver slices treated with 0.1% SDS for 3 days and sterilized with 0.1% acetic acid peroxide (FIG. 2A). After lyophilization, the LECM was solubilized and concentrated before addition to the culture medium. After the de-saturation process, hexagonal lobules, blood vessels, net-like microstructures, and collagen materials were observed (FIG. 2B), while the intracellular materials were efficiently removed from the LECM slices.
Approximately 99.3% ± 0.86% of total natural DNA was removed after de-saturation, while the amount of collagen and GAG remaining was 65.2% ± 17.3% (6.9 ± 1.2 μg / mg) and 102.6% ± 310 31.8% (591.7 ± 187.8 [mu] g / mg) (Fig. 2C). The antibody microarray showed that> 40 other liver-derived growth factors were preserved after de-saturation (Fig. 2D). LECM was 10-fold concentrated and added to the culture medium to 10% of the total volume of each differentiation step; The same amount of solubilized collagen was added to the medium as a negative control. AFP and ALB genes were expressed at higher levels in the LECM-treated group than in the control group, and were most effective when LECM was added only in the 4th step (Fig. 2E). Approximately two-fold increase in ALB expression in LECM-treated cells was detected by Western blotting (Fig. 2F). In addition, FACS analysis shows that ALB expression is highly expressed in LECM-treated cells (74.7% vs. 321 36.8%) than in the control (Figure 2G); However, AFP expression did not show any significant difference between the two groups. These results suggest that porcine iPSC-Hep can be a useful cell resource for the recalcified deglycosylated liver scaffold since their maturation efficiency is quantitatively and qualitatively improved in the LECM microenvironment.
In order to find the most efficient conditions for the differentiation of hepatic cells from porcine iPSC, the present inventors compared the differentiation ratios among the cell culture dish treatments used for hepatocyte differentiation in existing studies.
For the experimental group, the differentiation ratios were compared by comparing the degree of albumin gene expression between each group using the coating vessel quality and supporting cells (Gel: gelatin, Mat: Matrigel, Coll: Collagen, STO: STO feeder cell etc.
As a result, as shown in Fig. 3, hepatocyte differentiation of porcine iPSC was most favored in the Mat + Coll condition.
In addition, the present inventors confirmed the most effective conditions for liver differentiation by adding various concentrations of LECM to the cell culture medium under optimized differentiation conditions with the Mat + Coll coating. At this time, as described above with reference to FIG. 2E, LECM was added only to the 4th step of the hepatocyte differentiation protocol 1-4 of the present invention. Therefore, in
As a result, as shown in Fig. 4, when LECM was treated with 10% of the total volume of the medium, the expression of AFP and ALB was most effectively induced in iPSC-Hep (Fig. 4).
These results show that the method of the present invention can be efficiently applied to hepatocellular or organ specific differentiation.
Claims (11)
(a) forming and culturing embryoid bodies (EBs) by culturing degenerated stem cells in a culture vessel coated with Matrigel and collagen;
(b) inducing differentiation into hepatocytes by adding the differentiation inducing substances in the step (a) stepwise,
Wherein said differentiation inducing step comprises the steps of: (a) adding Y27362 and activin A; (B) adding bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF); (C) adding porcine hepatocyte growth factor (HGF) and insulin-transferrin-selenium X (ITS); And (d) adding OSM (oncostatin M), DEX (dexamethasone) and ITS (insulin-transferrin-selenium X); And
(c) Biocompatible solubilized scaffold concentrate derived from the defatted biomaterial at the induction of differentiation in step (b) is added to the medium at 10% of the total medium volume to enhance the differentiation rate of hepatocytes , ≪ / RTI &
The solubilized scaffold concentrate comprises
(c-1) decellularizing liver tissue with sodium dodecyl sulfate (SDS);
(c-2) obtaining an extracellular matrix (ECM) from the degummed tissue;
(c-3) lyophilizing the ECM of the step (c-2) and pulverizing the powder to form a powder;
(c-4) obtaining an acid-soluble extract obtained by adding an acid solution to the powder of the step (c-3) to obtain an acid-extracted extract;
(c-5) obtaining pepsin solubilized extract by the addition of pepsin to the acid-soluble extract of step (c-4);
(c-6) neutralizing the pepsin-solubilized extract of step (c-5) to obtain a soluble extract; And
(c-7) concentrating the soluble extract of step (c-6) to obtain a biocompatible solubilized scaffold concentrate.
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