Pluripotent stem cells transformed from human placental fibroblasts and induction method thereof

Abstract

The invention relates to the technical field of somatic cell reprogramming and cell transplantation therapy, in particular to a chemical induction method for reprogramming human placenta fibroblasts into pluripotent stem cells, which comprises the following steps: taking in-vitro human placenta fibroblasts, and reprogramming the in-vitro human placenta fibroblasts into induced epithelial-like cells under the action of TTNPB, calorphin, RepSox, Tranylcyromine and valproic acid; according to the method, the induced epithelial cells are subjected to continuous induction by utilizing a piripotent element, RepSox, TTNPB, Tranylcyromine, valproic acid, EPZ004777, AM580, CHIR99021 and DZNep, and finally, the induced pluripotent stem cells are obtained. According to the method, the reprogramming process only comprises two specific stages, the induction duration is greatly shortened, and the reprogramming cell induction efficiency is 0.0678% + / -0.0055%-0.0719% + / -0.0185%.

Description

Technical Field

[0001] The present invention relates to the technical field of somatic cell reprogramming and cell transplantation therapy, and particularly to pluripotent stem cells transformed from human placental fibroblasts and methods thereof. Background Art

[0002] Pluripotent stem cell technology occupies a crucial position in the field of regenerative medicine and is a solid foundation for treating major diseases. It provides new and highly potential technical means for a series of intractable diseases such as neurological diseases, cardiovascular diseases, diabetes, and retinal and corneal diseases. Embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), as the most critical types of pluripotent stem cells in the human body, have undeniable value in medical research and clinical applications. However, due to various considerations and limitations in the sources of acquisition, they have encountered many severe difficulties and challenges in clinical applications. This not only involves the complexity of approval but also problems at multiple levels such as technical difficulties in the acquisition process and potential risk factors, making the path of their clinical application full of uncertainties.

[0003] Induced pluripotent stem cells (iPSCs) are obtained by reprogramming somatic cells into a pluripotent state and have similar pluripotent differentiation potential to embryonic stem cells. iPSCs derived from somatic cells have the ability to differentiate into various functional cell types, providing a unique research model for exploring biological mechanisms. In clinical applications, the cells used for transplantation can be directly induced from the patient's somatic cells, reducing the risk of immune rejection. Therefore, induced pluripotent stem cells have a broader application prospect in clinical applications. In 2006, a research team led by Professor Shinya Yamanaka of Kyoto University in Japan first reported the successful induction of mouse fibroblasts into iPSCs by introducing four transcription factors (TFs), opening a new era in stem cell and regenerative medicine research. Although virus vector-mediated cell reprogramming is effective, it can cause permanent changes in the genome, generate heterogeneous iPSC cell lines, and increase the risk of tumor formation, posing a potential threat to the health of patients. Existing technologies also have methods for reprogramming and inducing pluripotent stem cells using small molecule drugs. iPSCs are obtained by culturing human umbilical cord mesenchymal stem cells in a chemically defined small molecule drug induction medium and a maintenance medium, and the induction time is 38 days. It can be seen that the existing technology has a relatively long overall culture cycle, thus restricting the improvement of culture efficiency. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a method and application for transforming human placental fibroblasts into pluripotent stem cells, effectively shortening the culture period to 12 to 16 days and improving the culture efficiency.

[0005] The first aspect of the present invention provides a method for inducing pluripotent stem cells transformed from human placental fibroblasts, comprising the following steps: Culturing ex vivo human placental fibroblasts in a first-stage induction medium to reprogram them into induced epithelial-like cells; Culturing the induced epithelial-like cells in a second-stage induction medium to obtain pluripotent stem cells; The first-stage induction medium comprises substances with the following final concentrations: adding 10 wt% serum replacement, 1 wt% N2 Supplement, 2 wt% B27A Supplement, 1 wt% GlutaMaxTM-I, 1 μM - 2 μM TTNPB, 10 μM - 25 μM forskolin, 8 μM - 10 μM RepSox, 10 μM Tranylcypromine, 0.5 mM valproic acid, and the solvent is a basal medium; The second-stage induction medium comprises substances with the following final concentrations: 10 wt% serum replacement, 1% N2 Supplement, 2 wt% B27A Supplement, 1 wt% GlutaMaxTM-I, 10 μM - 25 μM forskolin, 8 μM - 10 μM RepSox, 1 μM - 2 μM TTNPB, 10 μM Tranylcypromine, 0.5 mM valproic acid, 5 μM EPZ004777, 0.5 μM AM580, 5 μM - 10 μM CHIR99021, 0.1 μM DZNep; The basal medium is obtained by mixing equal volumes of DMEM / F-12 medium and Neurobasal medium.

[0006] The inventors of the present invention selected human placental fibroblasts as the starting material for reprogramming, which showed higher reprogramming responsiveness and retained the epigenetic memory of the early stage of embryonic development. By using two-stage induction culture, the stage characteristics of embryonic development could be successfully simulated, and the pluripotency quality of the terminal cells was significantly improved. The concentration of TTNPB in the first-stage induction medium and the second-stage induction medium was 1 μM to 2 μM. Compared with the addition amount of 5 μM to 10 μM in the prior art, it effectively reduced the occurrence of the problem of excessive activation of retinoic acid receptors leading to cell apoptosis. When the concentration of TTNPB was maintained at 1 μM to 2 μM, the optimal induction efficiency could be achieved, effectively promoting the second-stage induction process to develop in a positive direction. If the concentration was increased, it might cause signal transduction imbalance in the cells, have a toxic effect on the normal physiological functions of the cells, and thus affect the survival of the cells; conversely, if the concentration was decreased, the RAR and its downstream signal pathways could not be fully activated, resulting in a significant reduction in the induction efficiency, which was also not conducive to the normal differentiation of the cells and the achievement of the second-stage induction goal. And the sequential combination of forskolin and CHIR99021, that is, only forskolin was added to the first-stage induction medium, and both forskolin and CHIR9902 were added to the second-stage induction medium simultaneously, to achieve the synergistic activation of the cAMP and Wnt pathways. The entire culture cycle only required 12 days to 16 days, compared with 21 days to 28 days in the prior art, effectively shortening the culture time and improving the culture efficiency.

[0007] In another preferred embodiment, the time for culturing in the first-stage induction medium was 6 days to 8 days, and the first-stage induction medium was replaced every 2 days to 3 days during this period.

[0008] In another preferred embodiment, the temperature for culturing in the first-stage induction medium was 37 °C to 39 °C, the humidity was 90% to 95%, and the volume ratio of CO2 in the air was 5% to 5.5%.

[0009] In another preferred embodiment, the time for culturing in the second-stage induction medium was 6 days to 8 days, and the second-stage induction medium was replaced every 2 days to 3 days during this period.

[0010] In another preferred embodiment, the temperature for culturing in the second-stage induction medium was 37 °C to 39 °C, the humidity was 90% to 95%, and the volume ratio of CO2 in the air was 5% to 5.5%.

[0011] In another preferred embodiment, after culturing to obtain pluripotent stem cells in the second-stage induction medium, maturation culture was further included.

[0012] In another preferred embodiment, the specific process of the maturation culture was as follows: After culturing pluripotent stem cells in the first maintenance medium for 24 h to 48 h, transfer them to the second maintenance medium for culturing for 10 days to 12 days; The first maintenance medium is adding 10 μmol to 12 μmol of Y-27632 dihydrochloride to each liter of mTeSR TM 1 medium; the second maintenance medium is mTeSR TM 1 medium.

[0013] The second aspect of the present invention provides the pluripotent stem cells transformed from the human placental fibroblasts described above.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses human placental fibroblasts as the starting material for reprogramming, which has higher reprogramming responsiveness and retains the epigenetic memory in the early stage of embryonic development. By regulating the TTNPB concentration in the first-stage induction medium and the second medium to be 1 μM to 2 μM, not only the problem of cell apoptosis caused by over-activation of retinoic acid receptors is reduced, but also the second-stage induction process is promoted to develop in a positive direction, thereby accelerating the culture speed. And by adding only forskolin in the first-stage induction medium and adding forskolin and CHIR9902 simultaneously in the second-stage induction medium, the co-activation of the cAMP and Wnt pathways is achieved, further shortening the culture time. And with the cooperation of other small molecule compounds, the entire culture cycle only needs 12 days at the shortest, compared with 38 days in the prior art, the culture time is shortened by nearly 70%, greatly improving the culture efficiency. Description of the Drawings

[0015] Figure 1 It is a diagram of the induction process and cell morphology changes; in the figure, 1 and 2 are the morphological characteristics of fibroblasts: 1 is a 4-fold bright field image; 2 is a 10-fold magnified bright field image of HPFs within the white frame, 3 to 5 are the morphological characteristics of chemically induced human pluripotent stem cells. Specifically, 3 is a 4-fold bright field image of CihPSCs; 4 is a 10-fold magnified bright field image of CihPSCs within the white frame of 3; 5 is a 20-fold magnified bright field image of CihPSCs within the white frame of 4; in the figure, HPFs represents fibroblasts, and CihPSCs represents induced human pluripotent stem cells. Figure 1 It is a diagram of the morphological changes of cells involved in each stage of induction. Among them, 0D represents the morphological change diagram of cells at day 0, that is, the starting time, and the rest are the same; a represents the magnified image of the white-framed part in 5D, b represents the magnified image of the white-framed part in 7D, c represents the magnified image of the white-framed part in 16D, d represents the magnified image of the white-framed part in 24D, and e represents the magnified image of the white-framed part in d.

[0016] Figure 2 It is a diagram of the morphological changes of cells involved in each stage of induction. Among them, 0D represents the morphological change diagram of cells at day 0, that is, the starting time, and the rest are the same; a represents the magnified image of the white-framed part in 5D, b represents the magnified image of the white-framed part in 7D, c represents the magnified image of the white-framed part in 16D, d represents the magnified image of the white-framed part in 24D, and e represents the magnified image of the white-framed part in d.

[0017] Figure 3 It is a figure for reprogramming efficiency analysis results. Among them, A is a bright-field image of fibers and stem cells. Among them, 1 is the cell morphology image after reprogramming of HPFs, 2 is the enlarged image of the white frame line in 1, 3 is the cell morphology image after reprogramming of CihPSCs, and 4 is the enlarged image of the white frame line in 3; B is the quantitative figure of A.

[0018] Figure 4 It is a figure for identification and analysis of HPFs. Among them, A is a schematic diagram of the cell morphology of HPFs, 1 is the cell morphology image, and 2 is the enlarged image of the white frame line in 1; B is the karyotype analysis figure of HPFs, b1 represents the metaphase spread image of chromosomes, b2 represents the karyotype figure, where 1-22 are chromosome numbers, x is the x chromosome, and y is the y chromosome; C is the immunofluorescence detection of HPFs expression analysis figure.

[0019] Figure 5 It is a figure for identification and analysis of CiEP-Ls. Among them, A is the immunofluorescence detection of the expression analysis figure of the surface markers E-Cadherin, LIN28A, and KRT18 of CiEP-Ls, and B is the quantitative analysis result figure.

[0020] Figure 6 It is the figure of the expression results of fibroblast surface markers identified by Western blot in CiEP-Ls; A is the expression result figure, and B is the quantitative analysis figure of A. Among them, 1 is the quantitative analysis result figure of CD34, 2 is the quantitative analysis result figure of Vimentin, 3 is the quantitative analysis result figure of COL1A1, 4 is the quantitative analysis result figure of E-Cadherin, 5 is the quantitative analysis result figure of KRT18, and 6 is the quantitative analysis result figure of LIN28A.

[0021] Figure 7 It is the figure of the identification and analysis results of induced mature CihPSCs; A is the figure of the expression of surface markers identified by immunofluorescence in CihPSCs, and B is the quantitative analysis figure of A.

[0022] Figure 8 It is the figure of the expression levels of fibroblast surface markers identified by Western blot. A is the figure of the expression levels, and B is the quantitative analysis figure of A. Among them, 1 is the quantitative analysis figure of C-MYC, 2 is the quantitative analysis figure of E-Cadherin, 3 is the quantitative analysis figure of LIN28A, 4 is the quantitative analysis figure of Nanog, 5 is the quantitative analysis figure of SOX2, 6 is the quantitative analysis figure of OCT4, 7 is the quantitative analysis figure of SSEA1, 8 is the quantitative analysis figure of CD34, and 9 is the quantitative analysis figure of Vimentin.

[0023] Figure 9Figure showing the karyotype analysis results of CihPSCs and the results of CCK8 assay for identifying the proliferation and toxicity of induced cells; where A is the figure of karyotype analysis results, a1 is the metaphase spread of chromosomes, a2 is the karyotype map, in which 1-22 are chromosome numbers, x is the x chromosome, y is the y chromosome, and B is the figure of CCK8 assay for identifying the proliferation and toxicity of induced cells. Detailed implementation manners

[0024] The following is a detailed description of the specific implementation manners of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0025] Induced pluripotent stem cells are obtained by reprogramming somatic cells into a pluripotent state, and they have pluripotent differentiation potential similar to that of embryonic stem cells. iPSCs derived from somatic cells have the ability to differentiate into various functional cell types, providing a unique research model for exploring biological mechanisms under normal and pathological conditions. In clinical applications, the cells used for transplantation can be directly induced from the somatic cells of patients, reducing the risk of immune rejection. Therefore, induced pluripotent stem cells have a broader application prospect in clinical applications. In 2006, a research team led by Professor Shinya Yamanaka of Kyoto University in Japan first reported that mouse fibroblasts were successfully induced to transform into iPSCs by introducing four transcription factors (TFs), opening a new era in stem cell and regenerative medicine research.

[0026] Although viral vector-mediated cell reprogramming is effective, it can lead to permanent changes in the genome, generate heterogeneous iPSCs cell lines, and increase the risk of tumor formation, posing a potential threat to the health of patients. Reprogramming somatic cells into pluripotent stem cells using small molecule compounds has unique advantages. There are many types of small molecule compounds, with greater selectivity and higher safety. During reprogramming, relevant signaling pathways and the epigenetic modification state of cells can be regulated more flexibly and precisely, thus improving the efficiency of reprogramming induction. Therefore, small molecule compounds are widely used in the induction of pluripotent stem cells.

[0027] The present invention provides a chemical induction method for reprogramming human placenta fibroblasts into pluripotent stem cells using only small molecule compounds.

[0028] Example 1 1. Experimental materials Reagents: Non-Essential Amino Acids (NEAA), GlutamaxTM-I (100×), B27A Supplement, high-glucose DMEM medium, DMEM / F-12 medium, Neurobasal medium, 0.25% trypsin containing EDTA, fetal bovine serum (FBS), Knockout Serum Replacement, N2 Supplement (100×), Penicillin-Streptomycin, triple antibody (PSA) (100×), MEM-NEAA (100×), dimethyl sulfoxide (DMSO) were all purchased from Gibco. mTeSRTM1 Bassal Medium and mTeSRTM1 5X Supplement were purchased from Stem Cell. Valproic acid (VPA) and Triton X-100 were purchased from Sigma. CHIR99021, Repsox, Y-27632, Forskolin, TTNPB, Tranylcypromine, and DZNep were purchased from Selleckchem. EPZ004777 and AM580 were purchased from MCE. Donkey serum (NDS) and donkey anti-rabbit Cy3 were purchased from Jackson Lab. Donkey anti-mouse Alexa Fluor488 and anti-fluorescence quenching mounting medium were purchased from Invitrogen. Rb-Vimentin, Rb-CD34, and Rb-COL1A1 antibodies were purchased from BIOSS. Rb-Nanog antibody was purchased from Abcam. m-LIN28A, m-E-Cadherin, m-KRT18, Rb-SOX2, and Rb-OCT4 antibodies were purchased from Santa Cruz. Rb-SSEA1 antibody was purchased from Abclonal. Matrigel was purchased from Corning. DAPI staining solution was purchased from Beyotime Biotechnology Co., Ltd. Paraformaldehyde was purchased from West Asia Chemical Industry Co., Ltd., Shandong, China. All media and additives used in the present invention are of clinical grade.

[0029] High-glucose DMEM medium, DMEM / F12 medium, and Neurobasal medium were purchased from Gibco.

[0030] Media used in different stages: (1) Every 100 mL of normal culture of human placental fibroblasts contains the following components: high-glucose DMEM medium supplemented with 15 wt% serum, 1 wt% NEAA, 1 wt% GlutaMax TM-I (100×), and 1 wt% penicillin-streptomycin solution.

[0031] (2)Basal medium: DMEM / F-12 and Neurobasal with a volume ratio of 1:1.

[0032] (3)The first-stage induction medium is prepared by adding 10 wt% Knockout Serum Replacement, 1 wt% N2 Supplement (100×), 2 wt% B27A Supplement (100×), 1 wt% GlutaMaxTM-I (100×), 1 μM TTNPB, 25 μM Forskolin, 10 μM RepSox, 10 μM Tranylcypromine, and 0.5 mM VPA to every 100 mL of the basal medium.

[0033] (3)The second-stage induction medium is prepared by adding 10 wt% Knockout Serum Replacement, 1 wt% N2 Supplement (100×), 2 wt% B27A Supplement (100×), 1 wt% GlutaMaxTM-I (100×), 25 μM Forskolin, 10 μM RepSox, 1 μM TTNPB, 10 μM Tranylcypromine, 0.5 mM VPA, 5 μM EPZ004777, 0.5 μM AM580, 10 μM CHIR99021, and 0.1 μM DZNep to every 100 mL of the basal medium.

[0034] 2. Experimental methods In vitro culture of human placental fibroblasts: Human placental fibroblasts were inoculated into T25 culture flasks and cultured using the placental fibroblast maintenance medium. When the cells reached 80% confluence, they were digested with 0.25% trypsin, and passaged according to the cell quantity, from one flask to two or three flasks, and then continued to be cultured in a 37 °C carbon dioxide incubator.

[0035] Reprogramming of human placental fibroblasts into induced pluripotent stem cells (1)Induction of HPFs into epithelial-like cells (CiEP-Ls): HPFs in good condition within passage 3 were selected as the starting cells for induction and inoculated into 12-well plates at a density of 1.5×10 4 cells / well, and cultured in a 37 °C carbon dioxide incubator. After the cells grew to 80% confluence, they were induced in the first-stage induction medium for 8 days, with the medium changed every 2 days. The morphological changes of the cells were closely observed and photographed. The results are as follows Figure 1As shown by B in [reference], the morphological comparison between HPFs and CihPSCs visually demonstrated the significant changes in cell morphology during the induction process; specifically, HPFs exhibited typical monolayer adherent growth characteristics, with a long spindle-shaped cell morphology and obvious cytoplasmic extension; the induced CihPSCs formed characteristic three-dimensional cystic structures, spherical or ellipsoidal colonies. The culture conditions were adjusted and optimized in a timely manner according to the culture effect, and finally CiEP-Ls were obtained.

[0036] Among them, the induction medium in the first stage was prepared by mixing DMEM / F-12 and Neurobasal in a volume ratio of 1:1, and the induction medium contained the following substances: 10 wt% Knockout Serum Replacement, 1 wt% N2 Supplement (100×), 2 wt% B27A Supplement (100×), 1 wt% GlutaMax TM -Ⅰ (100×), 1 μmol / L TTNPB, 25 μmol / L Forskolin, 10 μmol / L RepSox, 10 μmol / L Tranylcypromine, 0.5 mmol / L VPA.

[0037] (2) Induction of CiEP-Ls into pluripotent stem cells (CihPSCs): Discard the old medium, add the second-stage induction medium to the induced CiEP-Ls and continue the induction for 6 to 8 days, and replace the fresh medium every 2 days during this period.

[0038] Among them, the induction medium in the second stage was prepared by mixing DMEM / F-12 and Neurobasal in a volume ratio of 1:1, and the induction medium contained the following substances: 10 wt% Knockout Serum Replacement, 1 wt% N2 Supplement (100×), 2 wt% B27A Supplement (100×), 1 wt% GlutaMax TM -Ⅰ (100×), 25 μM Forskolin, 10 μM RepSox, 1 μM TTNPB, 10 μM Tranylcypromine, 0.5 mM VPA, 5 μM EPZ004777, 0.5 μM AM580, 10 μM CHIR99021, 0.1 μM DZNep.

[0039] (3) Further maturation culture of CihPSCs: The old culture medium was discarded, the induced pluripotent stem cells were digested and re-plated, the cell colonies were transplanted onto the plates pre-coated with Matrigel, and the pluripotent stem cell maintenance medium was replaced.

[0040] Among them, the pluripotent stem cell maintenance medium is mTeSR TM 1 Bassal Medium, mTeSR TM 1 5XSupplement, 1% PSA (100×), the induced pluripotent stem cells were added to the mTeSRTM1 medium containing Y-27632 dihydrochloride (10μM) and cultured for 24 hours, and then cultured for another 10 days with fresh mTeSRTM1 medium without Y-27632 dihydrochloride, i.e. the induction was completed, and finally mature induced pluripotent stem cells were obtained.

[0041] 3. Experimental Results 1. Reprogramming of HPFs into CiEP-Ls The first stage is the chemical induction of epithelial-like cells. Human placental fibroblasts were induced to produce dense epithelial-like cells on the 8th day under the combined action of 1μM TTNPB, 25μM Forskolin, 10μM RepSox, 10μM Tranylcypromine, and 0.5mM VPA. In the initial stage, HPFs showed spindle-shaped or flat star-shaped morphological characteristics with slender protrusions. When the induction process begins, the initial cells will undergo obvious changes. Their original spindle shape gradually tends to be rounded, and the skeleton structure inside the cells also begins to rearrange. At the same time, the microvilli structure on the edge of the cells gradually appears. As the induction process advances to the middle stage, the cells further transform into a round shape. At this time, the adhesion molecules unique to epithelial cells begin to express. This change promotes the formation of tight junctions between cells, and the nuclear-cytoplasmic ratio of the cells changes, and the overall morphology of the cells becomes more three-dimensional. Until the late stage of induction, the cells transform into polygons, and the tight junctions are also fully formed. Through the gradual evolution of the above stages, the transformation process of HPFs to CiEP-Ls is finally achieved.

[0042] 2. Reprogramming of CiEP-Ls into CihPSCs The CiEP-Ls obtained in the first stage were replaced with the second stage induction medium containing 25 μM Forskolin, 10 μM RepSox, 1 μM TTNPB, 10 μM Tranylcypromine, 0.5 mM VPA, 5 μM EPZ004777, 0.5 μM AM580, 10 μM CHIR99021, and 0.1 μM DZNep, and the induction was continued for 6 to 18 days to obtain induced pluripotent stem cells, such as Figure 2 shown.

[0043] 3. Analysis of the induction efficiency of reprogramming HPFs into CihPSCs Based on the standardized experimental procedures, the conversion efficiency of small molecule compound combinations was systematically evaluated through three independent reprogramming experiments. After 16 consecutive days of induction in a standardized serum-free culture system, three fields of view were randomly selected through an inverted phase contrast microscope system. As Figure 3 shown, the number of CihPSCs and the total number of HPFs in the fields of view were accurately counted. The analysis results showed that the cell induction efficiency was 0.0678% ± 0.0055% - 0.0719% ± 0.0185% (mean ± SD, n = 9).

[0044] Example 2 Functional identification of the induced pluripotent stem cells obtained in Example 1 1) Identification and analysis of HPFs: HPFs were subjected to immunofluorescence staining for Vimentin, CD34, and COL1A1. Vimentin, CD34, and COL1A1 are all markers of fibroblasts. The immunofluorescence staining results showed that the induced initial cells could express the surface markers of fibroblasts and had obvious morphological characteristics of fibroblasts, such as Figure 4 shown in A and C of Figure 4 . The results of chromosome G-banding showed that HPFs were normal diploid cells without cross-contamination from cells of other species, as

[0045] shown in B of Figure 4 .

[0045] 2) Identification and analysis of CiEP-Ls: CiEP-Ls were subjected to immunofluorescence staining for E-Cadherin, LIN28A, and KRT18. E-Cadherin, LIN28A, and KRT18 are all markers of epithelial cells. The immunofluorescence staining results showed that CiEP-Ls obtained in the first stage simultaneously expressed E-Cadherin, LIN28A, and KRT18 and had obvious characteristics of epithelial cells, such as Figure 5 shown in A of Figure 5 . Detection by Western blot showed that the expression levels of the fibroblast surface markers CD34, Vimentin, and COL1A1 in CiEP-Ls were significantly reduced, while the protein expression levels of E-Cadherin, Lin28A, and KRT18 were significantly increased, as Figure 6 shown.

[0046] 3) Identification and analysis of induced mature CihPSCs The induced and matured CihPSCs were subjected to immunofluorescence staining for OCT4, C-MYC, Nanog, SSEA1, and SOX2. OCT4, Nanog, and SOX2 are all key transcription factors that are highly expressed in pluripotent stem cells and are crucial for maintaining pluripotency. Their expression levels are closely linked to the pluripotent state and are core markers for identifying pluripotency. C-MYC is highly expressed in both pluripotent stem cells and other proliferating cells, so it needs to be combined with the former three for pluripotency identification. SSEA1 is an important marker of stem cell pluripotency as Figure 7 shown. Western blot detection showed that the expression levels of the surface markers CD34 and Vimentin of fibroblasts were significantly reduced, while the surface markers E-Cadherin and Lin28A of epithelial cells were still stably expressed after induction and maturation. The expression levels of the surface markers OCT4, C-MYC, Nanog, SSEA1, and SOX2 of pluripotent stem cells increased significantly, as Figure 8 shown. To further detect the genetic characteristics of CihPSCs, this study performed karyotype analysis on the induced and matured cells. The results of chromosome G-banding showed that CihPSCs are normal diploid cells, as Figure 9 shown in A of Figure 9 . The CCK8 assay was used to evaluate the toxicity and proliferation effects of specific small molecules on cells. The detection results showed that within the entire detection concentration gradient range, the OD values of CihPSCs treated with small molecules showed only a very slight decrease compared with the control group (HPFs), and there was no statistically significant difference between each treatment group and the control group (P>0.05). The results indicated that under the conditions set in this experiment, this small molecule combination did not have an obvious inhibitory effect on the proliferation of HPFs cells, and the cell viability was not significantly affected. Furthermore, it can be inferred that this small molecule has no obvious toxicity to cells. From the perspective of cell proliferation, although there is a slight downward trend in numerical values, this change is not significant at the biological and statistical levels, indicating that this small molecule will not interfere with the normal proliferation process of cells due to cytotoxicity during the reprogramming induction process as

[0047] shown in B.

[0047] The above results indicate that the CihPSCs induced by the method in the present invention are extremely similar to pluripotent stem cells in key characteristics. At the molecular marker level, CihPSCs stably express a series of pluripotent stem cell-specific markers, such as the transcription factors OCT4, Nanog, and SOX2, etc. With the help of immunofluorescence technology, the specific localization and fluorescence intensity distribution of these markers in cells can be observed, intuitively indicating their expression characteristics; through Western blot analysis, specific bands corresponding to each marker can be detected, and their molecular weights and signal intensities are in line with the expected characteristics of pluripotent stem cell-related markers; from the perspective of cell morphology, the induced pluripotent stem cells show highly consistent morphological characteristics with pluripotent stem cells. The cells have round or oval cell nuclei, obvious nucleoli and clear structures, and the nuclear-cytoplasmic ratio is maintained at the level unique to pluripotent stem cells. The overall cell morphology is regular and has the typical appearance of pluripotent stem cells. Combining the above-mentioned multi-faceted analysis and detection results, it strongly confirms that the CihPSCs induced by the above-mentioned small molecule combination are highly similar to pluripotent stem cells in core dimensions such as molecular marker expression and cell morphology, and it can be determined that they have successfully possessed the key attributes and characteristics of pluripotent stem cells in essence.

[0048] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0049] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. An induction method for human placenta fibroblast-transformed pluripotent stem cells, characterized in that, Comprising the following steps: Culturing ex vivo human placental fibroblasts in a first-stage induction medium to recombine into induced epithelial-like cells; Culturing the induced epithelial-like cells in a second-stage induction medium to obtain pluripotent stem cells; The first-stage induction medium comprises substances with the following final concentrations: 10 wt% serum substitute, 1 wt% N2 Supplement, 2 wt% B27A Supplement, 1 wt% GlutaMaxTM-I, 1 μM - 2 μM TTNPB, 10 μM - 25 μM forskolin, 8 μM - 10 μM RepSox, 10 μM Tranylcypromine, 0.5 mM valproic acid, and the solvent is a basal medium; The second-stage induction medium comprises substances with the following final concentrations: 10 wt% serum substitute, 1% N2 Supplement, 2 wt% B27A Supplement, 1 wt% GlutaMaxTM-I, 10 μM - 25 μM forskolin, 8 μM - 10 μM RepSox, 1 μM - 2 μM TTNPB, 10 μM Tranylcypromine, 0.5 mM valproic acid, 5 μM EPZ004777, 0.5 μM AM580, 5 μM - 10 μM CHIR99021, 0.1 μM DZNep, and the solvent is a basal medium; The basal medium is obtained by mixing equal volumes of DMEM / F-12 medium and Neurobasal medium.

2. The induction method according to claim 1, wherein The culturing time in the first-stage induction medium is 6 days to 8 days, and the first-stage induction medium is replaced every 2 days to 3 days during this period.

3. The induction method according to claim 2, wherein The culturing temperature in the first-stage induction medium is 37°C to 39°C, the humidity is 90% to 95%, and the volume ratio of CO2 in the air is 5% to 5.5%.

4. The induction method according to claim 1, wherein The culturing time in the second-stage induction medium is 6 days to 8 days, and the second-stage induction medium is replaced every 2 days to 3 days during this period.

5. The induction method according to claim 4, wherein The culturing temperature in the second-stage induction medium is 37°C to 39°C, the humidity is 90% to 95%, and the volume ratio of CO2 in the air is 5% to 5.5%.

6. The induction method according to claim 1, characterized in that, After culturing in the second-stage induction medium to obtain pluripotent stem cells, maturation culturing is further included.

7. The induction method according to claim 6, wherein The specific process of the maturation culturing is as follows: Culturing the pluripotent stem cells in a first maintenance medium for 24 h to 48 h, and then transferring them to a second maintenance medium for culturing for 10 days to 12 days; The first maintenance medium is adding 10 μmol to 12 μmol of Y-27632 dihydrochloride into each liter of mTeSR TM 1 medium; the second maintenance medium is mTeSR TM 1 medium.

8. A pluripotent stem cell transformed from human placental fibroblasts obtained by the induction method according to any one of claims 1 to 7.

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