JP2005151907A5 - - Google Patents

Description

Placental or amniotic membrane-derived human stem cells, methods for establishing them, and methods for inducing differentiation into organs

  The present invention relates to placental or amniotic membrane-derived human stem cells (hereinafter sometimes simply referred to as human stem cells), a method for establishing the stem cells, and a method for inducing differentiation from the stem cells into organs. The human stem cells can be used, for example, as a material for organs for transplantation of patients suffering from various tissue or organ disorders or diseases.

The existence of stem cells exhibiting pluripotency is suggested, for example, in the hematopoietic system, intestinal tract, skeletal muscle, skin, or central nerve, and its existence is being demonstrated. For example, it is reported that neural stem cells can be transdifferentiated into all cell lineages (Non-patent Document 1), and bone marrow cells can be differentiated into all ectoderm cells, mesoderm cells, and endoderm cells as well. A report (Non-Patent Document 2) has also been made. On the other hand, there is an opposite report (Non-patent Document 3) that bone marrow cells hardly recognize multipotency, and knowledge about the multipotency of cell-related stem cells is complicated. In addition, cells with multipotency include embryonic stem cells, which have come to the spotlight as universal cells that open the way to human regenerative medicine and gene therapy, but may be born as humans From the ethical point of view, the method of destroying embryos and changing them into cultured cells is very problematic.
Therefore, the establishment of human stem cells that have multipotency and do not cause ethical problems is an urgent issue in Japan where organ transplantation medicine is behind.
Clark DL et al., “Science” (USA), 2000, 288, p. 660-1662 Jiang Y et al., “Nature” (UK), 2002, 418, p. 41-49 Wagers AJ et al., “Science”, (USA), 2002, Vol. 297, p. 2256-2259

  Accordingly, an object of the present invention is to provide a human stem cell capable of being passaged a sufficient number of times (30 times or more) as a stem cell from human placenta or amniotic membrane treated as a waste product during delivery, and further using the stem cell. The object is to provide a method for producing an organ.

The object is a human stem cell according to the invention, characterized in that it has the following cell biological characteristics:
(1) It is derived from human placenta or amniotic membrane.
(2) It has a normal diploid karyotype.
(3) Continue proliferation in an undifferentiated state.
(4) Alkaline phosphatase activity is positive.
(5) The transcription factors Rex-1, Oct-4, and Ecat-4 are expressed.
(6) Passage of 30 times or more is possible while maintaining multipotency.
Can be solved.
Further, the present invention is characterized in that cells obtained from human placenta or amniotic membrane are cultured using MEMα containing fetal bovine serum, epithelial cell growth factor, and leukemia inhibitory factor as a medium to form colonies. The present invention relates to a method for establishing human stem cells.
In the present invention, cells obtained from human placenta or amniotic membrane are cultured using MEMα containing fetal bovine serum, epithelial cell growth factor, and leukemia inhibitory factor as a medium to form colonies. a) Karyotype, (b) Proliferation in undifferentiated state, (c) Presence or absence of alkaline phosphatase activity, (d) Expression of Rex-1, Oct-4, and Ecat-4, and (e) Multiple differentiation The present invention relates to a method for establishing human stem cells, characterized by screening using the presence or absence of ability as an index.
The present invention also relates to a human transformant that can be obtained by introducing a foreign gene into the human stem cell.
The present invention also relates to a human differentiated cell, a human differentiated tissue, or a human organ derived from the human stem cell or the human transformant.
The present invention also relates to a human-animal chimeric embryo, chimeric fetus, or chimeric individual derived from the human stem cell or the human transformant.
The present invention also relates to a method for producing an organ, characterized by using the human stem cell or the human transformant.
Furthermore, the present invention provides a method of transplanting stem cells differentiated into bone marrow cells to suppress rejection, simultaneously with organ cells differentiated for transplantation.
For example, a method of suppressing rejection by simultaneously transplanting human stem cells that have been differentiated until spontaneous pulsation in the same myocardial infarction patient and stem cells that have been differentiated into bone marrow cells. provide.

  The placenta or amniotic membrane-derived human stem cells of the present invention are used as research materials for molecular biology, embryology, biochemistry, etc. for elucidating the mechanism of transdifferentiation in vivo or in vitro as stem cells, or organ transplantation The differentiation induction of the present invention is extremely valuable for regeneration of various medical cells, tissues, or organs using an in vitro culture system. Moreover, by creating a chimeric embryo from a transgenic human stem cell and an animal embryo, it is also useful as a donor cell for production of a genetically modified animal that becomes a bioreactor of a useful drug.

The human stem cells of the present invention have the following cell biological characteristics:
(1) It is derived from human placenta or amniotic membrane.
(2) It has a normal diploid karyotype.
(3) Continue proliferation in an undifferentiated state.
(4) Alkaline phosphatase activity is positive.
(5) The transcription factors Rex-1, Oct-4, and Ecat-4 are expressed.
(6) Passage of 30 times or more is possible while maintaining multipotency.

  The human stem cells of the present invention are derived from human placenta or amniotic membrane. The term “placenta” as used herein refers to a portion of the fetal membrane where the choroid (or capsule) villi joins the maternal endometrium, and the choroid belonging to the fetus is called the placenta. In the present specification, the “amniotic membrane” is a transparent thin film that directly covers the innermost fetus in the fetal membrane, wraps the umbilical cord, and is covered with the allantoic membrane on the outside. Some are filled with amniotic fluid and the fetus is floating in the amniotic fluid. Thus, the fetal membrane is composed of choroid, allantoic membrane, and amniotic membrane, and is mainly derived from the embryonic ectoderm and the upper blastoderm from the inner cell mass.

The human stem cells of the present invention have a normal diploid karyotype (ie, 46XX or 46XY).
Further, the human stem cells of the present invention can be passaged 30 times or more (preferably 40 times or more, more preferably 50 times or more, and further preferably 60 times or more) while maintaining an undifferentiated state and multipotency. is there.
In the present specification, “passaging” means that a part of cells in a cell culture container that has reached a confluent state (for example, 1/5 to 1/10) is substantially the same as another cell. This means transferring to a culture vessel and again allowing the cells to grow to a confluent state, and this series of operations is defined as one passage. Note that each cell can undergo about 5 cell divisions in a single pass.

Whether or not the human stem cells of the present invention are maintained in an undifferentiated state is determined by, for example, various known undifferentiated markers established in mammals (for example, mice or cows) (for example, JP-A-2002-176974). Can be confirmed. Examples of the undifferentiated marker include positive alkaline phosphatase activity or expression of transcription factors Oct-4, Rex-1, and Ecat-4.
Alkaline phosphatase activity and transcription factor Oct-4 expression can be confirmed, for example, by the method described in JP-A No. 2002-176773. For expression of transcription factor Rex-1, for example, Jean Wye ( Jiang Y) et al., “Nature” (UK), 2002, 418, p. The expression of the transcription factor Ecat-4 can be confirmed by, for example, Mitsui K. et al., “Cell”, (USA), 2003, Vol. 113. , P. 631-642.

Whether or not the human stem cell of the present invention has multipotency is determined by, for example, various known confirmation methods (for example, JP-A-2002-176972) established in mammals (for example, mice or cows). Can be confirmed.
As a method for confirming pluripotency, for example, differentiation into blood progenitor cells, nerve cells, or hepatocytes in a culture system, and Flk-1 (vascular endothelial marker), CD45 or CD34 (blood cell marker), GFAP (glial fibrous acid) protein (astrocyte marker), nestin (neural stem cell marker), albumin or cytokeratin 18 (stem cell marker), or a method for confirming pluripotency by staining with an antibody against human stem cells An enhanced green fluorescent protein (EGFP) gene, which is a gene, is introduced, and EGFP gene-introduced hepatocytes are transplanted into the subcutaneous tissue of a congenital thymus-deficient mouse, and the presence or absence of teratocarcinoma formation ability and the formed carcinoma are histologically examined. How to confirm by analysis Etc. can be mentioned.
Furthermore, for example, an EGFP gene, which becomes a marker gene, is introduced into the human stem cell of the present invention, a chimeric embryo is prepared from the EGFP-introduced human stem cell and a non-human animal embryo, and the chimeric embryo is transferred to an in vitro culture or a foster parent. After development, by examining the cultured chimeric embryo or transplanted chimeric fetus with a fluorescence microscope, and examining the contribution state of the EGFP gene-introduced human stem cells in the chimera embryo, it is possible to confirm multipotency it can.

  The human stem cell of the present invention can be established by, for example, the human stem cell establishment method of the present invention, that is, by culturing cells obtained from human placenta or amniotic membrane in a predetermined medium to form colonies. For example, the human placenta or amniotic membrane removed aseptically can be mechanically pre-chopped and then dissociated to each cell by protease (for example, trypsin) treatment, and then used for the following culture.

  In the human stem cell establishment method of the present invention, a medium in which fetal bovine serum (FCS), epithelial cell growth factor (EGF), and leukemia inhibitory factor (LIF) are added to a MEMα medium (hereinafter referred to as an establishment medium). ). The amount of each component added to the establishment medium can be, for example, 5 to 10% for FCS, 10 to 50 ng / mL for EGF, and 10 to 50 ng / mL for LIF.

Tables 1 and 2 show the composition of the MEMα medium. The unit of each numerical value is “mg / L”.

In the establishment method of the present invention, colonies are formed by culturing on an appropriate culture vessel (for example, a plastic 4-well dish) using the establishment medium. As the culture conditions, general culture conditions used for culturing normal animal cultured cells can be applied as they are, for example, conditions of 36.5 to 37.5 ° C. (preferably 37 ° C.) and 5% CO ₂ . Cultivation can be carried out by crystallization.
In the primary culture, mesenchymal cell colonies having a bipolar morphology appear 3 to 4 days after the start of the culture. After reaching confluence in 7 to 10 days from the start of culture, the cells are detached from the medium by trypsin treatment, and the detached cells are dispersed into individual cells, and then the medium used in the primary culture (that is, the medium for establishment) is used. Re-culture. A few cells (diameter 30-60 μm) having a bipolar or multipolar morphology different from fibroblasts appear in the cultured cells, but these cells are removed using a pipette and are not separated by trypsin. Transfer to a new culture dish and continue culturing until confluent under the same conditions as the primary culture. This cell having the feature of dividing once a day is a pluripotent human stem cell capable of differentiating into various cell tissues such as nerve, skin and digestive tract by changing the culture conditions.

  Regarding the obtained cells, (a) karyotype, (b) growth in an undifferentiated state, (c) presence or absence of alkaline phosphatase activity, (d) expression of Rex-1, Oct-4, and Ecat-4 And (e) analyzing the presence or absence of pluripotency to confirm that the obtained cells are the human stem cells of the present invention. That is, (a) having a normal diploid karyotype, (b) continuing to grow in an undifferentiated state, (c) being positive for alkaline phosphatase activity, (d) transcription factors Rex-1, Oct -4 and Ecat-4 are expressed, and (e) the human stem cell of the present invention is capable of being passaged 30 times or more while maintaining multipotency.

A preferred embodiment in the establishment method of the present invention is:
(I) A cell colony having a bipolar or multipolar morphology by subculturing cells obtained from human placenta or amniotic membrane using MEMα containing fetal bovine serum, epidermal growth factor, and leukemia inhibitory factor as a medium Obtaining a step,
(Ii) taking out the cell mass, culturing MEMα containing fetal bovine serum, epidermal growth factor, and leukemia inhibitory factor as a medium to obtain colonies composed of cells having a spherical or elliptical shape, and ( iii) Regarding the cells obtained in the above step (ii), (a) karyotype, (b) proliferation in an undifferentiated state, (c) presence or absence of alkaline phosphatase activity, (d) Rex-1, Oct-4 And ecat-4 expression and (e) the step of analyzing the presence or absence of pluripotency.

  The present invention includes human transformants (that is, gene-transferred cells) that can be obtained by introducing any desired foreign gene into the human stem cells of the present invention. Examples of the gene introduction method to the human stem cell of the present invention include a known gene introduction method, for example, a method using a commercially available gene introduction reagent [for example, FuGene (Roche) or Effectene (Qiagen)].

  Further, the present invention includes differentiated cells, differentiated tissues, or organs derived from the human stem cells or transformants of the present invention. Examples of the tissue or organ include nerves, muscles (for example, cardiac muscle or skeletal muscle), digestive tract, liver, pancreas, skin, blood cells, bone marrow, blood vessels, and the like. The differentiated cell, differentiated tissue, or organ of the present invention can be obtained by appropriately selecting a known differentiation method according to the purpose except that the human stem cell or transformant of the present invention is used.

Furthermore, the present invention includes a human-animal (eg, mammal, eg, mouse, rat, cow, horse, pig, monkey) chimeric embryo, chimeric fetus, or chimera derived from the human stem cell or transformant of the invention. Individuals are included. The chimera embryo, chimera fetus, or chimera of the present invention can be obtained by appropriately selecting a known production method according to the purpose other than using the human stem cell or transformant of the present invention. .
For example, by creating a chimeric embryo in a human stem cell or transformant and non-human animal embryos it was introduced genes, transplanted chimeric embryos cultured in vitro or foster mothers development, culture chimeric embryo, chimeric fetuses , or it can be obtained chimeric individual body.
More specifically, for example, 10 to 15 human stem cells are injected into the blastomere in the 8-16 cell stage fertilized embryo or the space between the embryonic space and the chimeric embryo to prepare a chimeric embryo. A chimeric individual can be obtained by developing it into a blastocyst and transplanting it into the uterus of a foster parent animal.

The present invention includes various methods using the human stem cells of the present invention. For example, the present invention includes a method of transplanting stem cells that have been differentiated into bone marrow cells to suppress rejection, simultaneously with organ cells that have been differentiated for transplantation. For example, it is possible to suppress rejection by simultaneously transplanting human stem cells that have been differentiated to spontaneous pulsation in the same myocardial infarction patient and stem cells that have been differentiated into bone marrow cells. .
In the method of the present invention, for example, in order to induce regeneration of cardiomyocytes and vascular endothelium against coronary artery necrosis, the stem cells of the present invention are injected into the ventricle, for example, by intravenous injection or catheter. Administration can be performed. The cell dose can be, for example, 1,000 to 100,000 (for example, JP 2002-507407 A).

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1: Acquisition of placenta or amniotic membrane-derived human stem cells The human neonatal placenta and amniotic membrane removed aseptically by caesarean section were cut into about 0.5-1 cm ² with a sterile surgical scissors, respectively, and 70% After spraying with ethanol and sterilizing with flame, antibiotic-added PBS (-) (without Dulbecco, Ca and Mg) (penicillin 2000 units / mL; Meiji Seika, streptomycin 100 μg / mL; Meiji Seika, and Fungizone 25 μg / mL Washed several times with Gibco BRL). The sample is further chopped to about 1 to ² mm ² on a sterile petri dish, and then 10% at 37 ° C. and 5% CO _{2 in} a drop of 0.25% trypsin and 0.1% EDTA (Gibco BRL) aqueous solution. Incubated for 1 minute. The trypsin solution containing this sample was diluted with 5 mL of the PBS (−), centrifuged (1000 rpm, 5 minutes), the precipitate was suspended in 5 mL of PBS (−), and then centrifuged again under the same conditions. .

The resulting precipitate contained 10% FCS (Gibco BRL), antibiotics and antibacterial agents (penicillin, streptomycin, and fungizone), and further added 10 ng / mL-EGF (Sigma), 10 ng / mL-LIF (Sigma). Incubation was continued under conditions of 37.0 ° C. and 5% CO ₂ on a 4-well dish (Nunc) in which 500 μL of MEMα (Gibco BRL) was added to each well.
A mesenchymal cell having a bipolar morphology appeared as a colony around or in the well of the sample minced after 3 to 4 days of culture. The culture was further continued for about 4 to 7 days. When the cells reached confluence, they were treated with a trypsin solution for about 6 to 7 minutes, and then the cells were dissociated using a pipette. After dissociation, the sample was diluted with PBS (−), washed twice by centrifugation, and the resulting precipitate was plated on a 4-well dish at a concentration of 1/4 to 1/5 under the same culture conditions as described above ( 1st passage).

A few cells (diameter 30-60 μm) having a bipolar or multipolar morphology different from fibroblasts appeared in the cultured cells. These cells were taken out using a pipette, transferred to a new culture dish without being separated with trypsin, and transferred to a new culture dish, and the culture was continued until it became confluent under the same conditions as the primary culture. Screening and passaging of these cells with various markers as ES cells were performed according to the procedures described in JP-A No. 2002-176773, thereby establishing a human undifferentiated stem cell line (Ham1 to Ham8). ). Among these, Ham1 and Ham4 to Ham8 are derived from amniotic membrane, and Ham2 and Ham3 are derived from placenta.
Specifically, as undifferentiated markers, (1) alkaline phosphatase activity was positive, (2) transcription factors Oct-4, Rex-1, and Ecat-4 were confirmed and passaged. It was confirmed that he was in normal karyotype (46XX or 46XY) in his 20s. Regarding the possible number of passages, it was confirmed that passage was possible up to at least 60 generations.

For multipotency, placenta or amnion-derived human stem cells (Ham 1-8) of passage 20 were 10 in MEMα medium containing EGF, FGF (fibroblast growth factor) 2 and FGF9 at a concentration of 20 ng / mL, respectively. As a result of culturing for 30 to 30 days, neurons that showed an antibody positive reaction with GFAP antibody and nestin antibody, which are astrocyte markers and neural stem cell markers, were detected, respectively.
Furthermore, by culturing human stem cells (Ham 1 to 8) in a MEMα medium containing vascular endothelial growth factor (VEGF) at a concentration of 50 ng / mL and supplemented with 10% FCS for about 14 days, a phenotype of the vascular endothelial system is obtained. And cell colonies showing blood cell phenotypes appeared, and positively reacted with the vascular endothelial marker Flk-1 antibody, blood cell markers CD45 antibody and CD31 antibody, respectively. Further, by culturing human stem cells (Ham1 to Ham8) in a MEMα medium containing FGF4 at 20 ng / mL and hepatocyte growth factor (HGF) at a concentration of 20 ng / mL and supplemented with 10% FCS, about 50% is obtained. Hepatocyte phenotype positive for albumin antibody and cytokeratin 18 antibody.
Various antibodies and cell growth factors were purchased from Sigma.
From the above results, it was proved that placenta or amnion-derived human stem cells have multipotency in the culture system.

Example 2: Gene transfer to placenta or amnion-derived human stem cells In this example, gene transfer to the human stem cells of the present invention was performed.
The DNA fragment used for gene transfer was prepared by the following procedure. That is, a gene vector containing a cytomegalovirus enhancer, a chicken β-actin promoter, a rabbit β globin sequence, a neomycin resistance gene, and an enhanced green fluorescent protein (EGFP) cDNA is subcloned by a conventional method and then digested with PVUI / HindIII. The DNA fragment thus obtained was separated from the vector by 1% agarose gel electrophoresis and purified. The purified gene fragment was dissolved in a 10 mmol / L-Tris-HCl / 10 mmol / L-EDTA solution to a concentration of 1 μg / μL and stored frozen until used for gene transfer.

  Using human stem cells (Ham-1) established in Example 1, human stem cells with passage number 15 are freeze-thawed, seeded in one 4-well dish (Nunc), and lipid-based commercial gene transfer Gene introduction into human stem cells was performed by co-culture of the reagent (FuGene; Roche) and the gene fragment.

That is,
(1) Inject 80 μL of serum-free MEMα medium into a sterilized test tube,
(2) Add 5 μL of FuGene, mix well,
(3) Add 2 μL of DNA to the mixture, mix well and incubate at room temperature for about 30 minutes.
(4) For gene transfer treatment, take 20 μL of DNA-FuGene mixture and add the same amount to each well containing cultured human stem cells,
(5) Continue culturing for at least 24 hours in a medium supplemented with DNA-Reagent mixed solution for a maximum of 48 hours,
(6) Subsequently, only transgenic human stem cells were selected by culturing in an FCS-containing MEMα medium containing neomycin antibiotic G418 at a concentration of 400 μg / mL for 7 to 10 days. Furthermore, after switching to a normal medium and repeated passages (passage number = 17 to 60), the transgenic human stem cells were cryopreserved.

Example 3 In Vivo Transplantation of Human Stem Cells into Congenital Athymic Mice (ie, Nude Mice) In order to confirm the ability of human stem cells to form tumors in immunodeficient nude mice, 6-8 week old nude mice Approximately 5-8 × 10 ⁶ pieces were injected into the subcutaneous tissue using 25G needles. When the mice were sacrificed 2 months later, tumor formation was observed in 8 out of 10 transplanted animals. After excising them, all tissue specimens were prepared and examined histologically. In these specimens, tissues of all three germ layers (glia, bone, epithelium-like tissue, and gastrointestinal tract) were formed, and the multipotency of human stem cells was confirmed. Moreover, the same result was able to be obtained by implementing the same process also about the placenta or amniotic membrane origin human stem cell Ham2-8 established in Example 1. FIG.

  The human stem cells of the present invention can be used, for example, as a material for organs for transplantation of patients suffering from various tissue or organ disorders or diseases.