Cell type used for producing induced pluripotent stem (iPS) cells and preparation method and application thereof

Abstract

The invention discloses four cell types which have wide heteroplastic transplantation application prospect and can be effectively induced into induced pluripotent stem (iPS) cells. The origins of three cell types are placental tissue, namely amnion mesenchymal cell, chorion mesenchymal cell and umbilical cord mesenchymal cell; and the origin of the other one is amniotic fluid cell. Besides, the invention also discloses an induction reprogramming method for producing cells capable of producing induced pluripotent stem (iPS) cells by efficient induction, including the following steps: cDNA containing pluripotent stem cell factor is respectively introduced into the four primary culture cells; the four primary cells in which cDNA is introduced are respectively cultured on appropriate culture mediums, primary iPS is obtained and then quality of iPS is optimized on appropriate culture mediums, and cloning of pluripotent stem cell can be primarily evaluated. In the invention, three mesenchymal cells of placenta origin and amniotic fluid cell all can be induced into iPS, wherein the chorion mesenchymal cell is compared with fibroblast, efficiency of induction reprogramming is improved by over 100 times, efficiency is about 2.3%, and the efficiency is slightly higher than that of horny cell; and a means which is more effective and more pertinent is provided for building disease model, screening drug and controlling oriented differentiation.

Description

technical field

[0001] The invention relates to the field of cells, in particular to four cell types that can be induced to produce induced pluripotent stem cells (iPS) and have the prospect of allogeneic transplantation, their preparation method and application. Background technique

[0002] Stem cells (stem cells) are the initial source of the human body and its various tissue cells, and its most notable biological characteristics are the ability of self-renewal and continuous proliferation, as well as the potential of multidirectional differentiation. Stem cells are divided into adult stem cells (somaticstem cells) and embryonic stem cells (embryonic stem cells, ES cells) according to different sources. Adult stem cells include bone marrow mesenchymal stem cells, pancreatic stem cells, neural stem cells, etc., which exist in adult tissues.

[0003] In 1981, the isolation and cultivation of ES cells was first successfully done in mice, which is the most widely studied and...

Examples

Embodiment 1

[0055] Cell Preparation and Culture

[0056] For amniocytes, a certain amount of amniotic fluid is obtained by amniocentesis of pregnant women undergoing prenatal care. The precipitate obtained after centrifugation was resuspended in the amniotic fluid medium for culture for 3-4 days, and then replaced with fresh amniotic fluid medium to continue the culture.

[0057] For the three kinds of cells derived from placenta, the placental tissue and umbilical cord isolated from normal full-term cesarean section waste were taken under sterile conditions, washed several times with 1×PBS, and the amnion and chorion were peeled off with tweezers and placed in PBS separately Then use surgical scissors to cut the amnion and chorion into about 1mm 3small organizational blocks. (1) In order to obtain amniotic mesenchymal cells, trypsin was added to amniotic membrane fragments to digest at 37°C for 30-45 minutes, a total of 3 times, then added Dispase at a final concentration of 1.2U / ml, d...

Embodiment 2

[0062] Viral vectors infect three types of cells derived from placental tissue and amniocytes

[0063] According to the method described in Example 1, in the p6 well culture plate, approximately 4 × 10 4 Each cell was inoculated with three kinds of cells derived from human placenta, and about 8×10 cells per well for amniocytes 4 Cells were seeded at 37°C, 5% CO 2 Cultured overnight under normal culture conditions (such as figure 1 ), and the collected virus supernatants were used to infect three cultured placenta-derived cells and amniocytes respectively. The viral supernatant was obtained by transfecting 293T cells (Lipofectamine 2000, Invitrogen Corporation) with a retroviral pMX vector (Addgene Corporation) containing human Oct4, Sox2, Klf4 and c-Myc cDNA according to conventional methods ( See Sambrook, Fritsch and Maniatis, A Laboratory Guide to Molecular Cloning, 3rd Edition (2002)).

Embodiment 3

[0065] Continued culture of infected cells and clone selection

[0066] On the second day after infection, the culture medium of the three kinds of placenta-derived cells and amniocytes after infection was replaced with the DFBS medium described in Example 1, and the 2 Cultivate for 4 days under normal culture conditions, replace the medium once a day, digest it into a single cell suspension with 0.25% trypsin-EDTA (Gibco) at 37°C, and divide the digested cells at a density of about 10,000 cells per culture dish Inoculated into 100 mm culture dishes, which were pre-coated with trophoblast cells as described in Example 1. The seeded cells were incubated at 37°C, 5% CO 2 Under conventional culture conditions, cultured between 15-30 days, ES-like clones can be seen under microscope (such as figure 2 Shown are chorionic mesenchymal cells forming typical hES-like clonal morphology on trophoblast 28 days after infection with SKOM). The hES-like clones were picked and propagated ...

technical field

[0001] The invention relates to the field of cells, in particular to four cell types that can be induced to produce induced pluripotent stem cells (iPS) and have the prospect of allogeneic transplantation, their preparation method and application. Background technique

[0002] Stem cells (stem cells) are the initial source of the human body and its various tissue cells, and its most notable biological characteristics are the ability of self-renewal and continuous proliferation, as well as the potential of multidirectional differentiation. Stem cells are divided into adult stem cells (somaticstem cells) and embryonic stem cells (embryonic stem cells, ES cells) according to different sources. Adult stem cells include bone marrow mesenchymal stem cells, pancreatic stem cells, neural stem cells, etc., which exist in adult tissues.

[0003] In 1981, the isolation and cultivation of ES cells was first successfully done in mice, which is the most widely studied and...

Examples

Embodiment 1

[0055] Cell Preparation and Culture

[0056] For amniocytes, a certain amount of amniotic fluid is obtained by amniocentesis of pregnant women undergoing prenatal care. The precipitate obtained after centrifugation was resuspended in the amniotic fluid medium for culture for 3-4 days, and then replaced with fresh amniotic fluid medium to continue the culture.

[0057] For the three kinds of cells derived from placenta, the placental tissue and umbilical cord isolated from normal full-term cesarean section waste were taken under sterile conditions, washed several times with 1×PBS, and the amnion and chorion were peeled off with tweezers and placed in PBS separately Then use surgical scissors to cut the amnion and chorion into about 1mm 3small organizational blocks. (1) In order to obtain amniotic mesenchymal cells, trypsin was added to amniotic membrane fragments to digest at 37°C for 30-45 minutes, a total of 3 times, then added Dispase at a final concentration of 1.2U / ml, d...

Embodiment 2

[0062] Viral vectors infect three types of cells derived from placental tissue and amniocytes

[0063] According to the method described in Example 1, in the p6 well culture plate, approximately 4 × 10 4 Each cell was inoculated with three kinds of cells derived from human placenta, and about 8×10 cells per well for amniocytes 4 Cells were seeded at 37°C, 5% CO 2 Cultured overnight under normal culture conditions (such as figure 1 ), and the collected virus supernatants were used to infect three cultured placenta-derived cells and amniocytes respectively. The viral supernatant was obtained by transfecting 293T cells (Lipofectamine 2000, Invitrogen Corporation) with a retroviral pMX vector (Addgene Corporation) containing human Oct4, Sox2, Klf4 and c-Myc cDNA according to conventional methods ( See Sambrook, Fritsch and Maniatis, A Laboratory Guide to Molecular Cloning, 3rd Edition (2002)).

Embodiment 3

[0065] Continued culture of infected cells and clone selection

[0066] On the second day after infection, the culture medium of the three kinds of placenta-derived cells and amniocytes after infection was replaced with the DFBS medium described in Example 1, and the 2 Cultivate for 4 days under normal culture conditions, replace the medium once a day, digest it into a single cell suspension with 0.25% trypsin-EDTA (Gibco) at 37°C, and divide the digested cells at a density of about 10,000 cells per culture dish Inoculated into 100 mm culture dishes, which were pre-coated with trophoblast cells as described in Example 1. The seeded cells were incubated at 37°C, 5% CO 2 Under conventional culture conditions, cultured between 15-30 days, ES-like clones can be seen under microscope (such as figure 2 Shown are chorionic mesenchymal cells forming typical hES-like clonal morphology on trophoblast 28 days after infection with SKOM). The hES-like clones were picked and propagated ...

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