Serum-free culture method for converting hUC-MSCs into hiPSCs through small molecule compound induction

Abstract

The invention provides a serum-free culture method for converting hUC-MSCs into hiPSCs through induction of a small molecule compound, can be used for preparing pluripotent stem cells, and belongs to the field of biomedicine. Comprising thymine analogs, cAMP activators, TGF-beta receptor inhibitors, bone morphogenetic proteins, RA receptor activators, GSK3 inhibitors and basic fibroblast growth factors, and the culture system does not contain serum. And 2, in the process of carrying out micromolecule compound reprogramming on the hUC-MSCs by utilizing the method provided by the invention, frequent digestion, inoculation and tray separation on the hUC-MSCs are not needed, so that compared with the existing culture method, the operation steps are simplified, the cell loss generated in the digestion, inoculation and tray separation process is reduced, and the cell quality is improved. According to the invention, the reprogramming efficiency of the small molecule compound on hUC-MSCs is obviously improved; and 3, by detecting the co-expression (such as OCT4, SOX2 and NANOG) of transcription factors in reprogrammed cells of a small molecule compound reprogrammed pluripotent stem cell line obtained by the technology, it is proved that the hiPSCs obtained by inducing the hUC-MSCs have stable pluripotency capable of continuous passage.

Description

Technical Field

[0001] The present invention provides a serum-free culture method for inducing hUC-MSCs to be converted into hiPSCs by using small molecule compounds, which can be used for preparing pluripotent stem cells and belongs to the field of biomedicine. Background Art

[0002] Cells are the basic units that make up the structure and function of the human body. An adult human body has 40 trillion to 60 trillion cells. These cells can be divided into about 200 types, and different types of cells have different functions. All cells in the human body originally come from the same cell, the fertilized egg. So, can humans obtain "seed" cells in vitro that are similar to fertilized eggs and can differentiate into all types of cells? In 1981, Evans et al. successfully isolated a type of cell from early mouse embryos and named it embryonic stem cells. However, if embryonic stem cell technology is to be applied clinically, two problems must be solved: (1) Immune rejection between different individuals limits its application to a certain extent. (2) More importantly, the acquisition of embryonic stem cells will destroy the embryo, which makes the application of embryonic stem cells a major ethical controversy.

[0003] Cell differentiation is the core event of organism development. Cell differentiation can form terminally differentiated cells with various mature functions, and the high stability of the morphological structure and function of terminally differentiated cells is the basis of the life activities of the organism. However, the differentiation state of terminal cells is not completely fixed and unchangeable. Under certain special conditions, differentiated cells can return to an undifferentiated state, which is called dedifferentiation. The cell fate state refers to the relatively stable existence form of its genetic information and contents, which is affected by both external and internal factors. Changing the internal factors of the cell or the environment in which it is located is the basis for achieving the transformation of different cell fate states. Cell programming and reprogramming are the processes in which the cell fate state changes. For example, the core network of transcription factors, epigenetic modification, cell metabolism and signal regulation have all undergone corresponding changes, so changing any of the above processes can regulate the cell fate state. Among them, pluripotent stem cells have the characteristics of unlimited proliferation and the ability to differentiate into all functional cell types of the body. These magical qualities make them have a wide range of application value in the fields of cell therapy, drug screening and disease models, and are the most critical "seed cells" in the field of regenerative medicine. However, how to reverse this natural development process and make highly differentiated adult cells regain a pluripotent state similar to embryonic stem cells has always been one of the most important scientific issues in the field of stem cell and regenerative medicine research. The field of life sciences has been working towards this goal for decades. In 2006, Yamanaka et al. transferred four transcription factors (Oct4, Sox2, Klf4, cMyc) into mouse fibroblasts through retroviral vectors to turn them into multipotent stem cells, thus creating iPS technology. This technology proved that mammalian somatic cells can be reversed to the early state of embryonic development through reprogramming and regain "pluripotency", and won the Nobel Prize in Physiology or Medicine in 2012. The establishment of iPS technology has broken the ethical restrictions of traditional ESCs, provided a new method for constructing patient-specific stem cell lines, and greatly accelerated the process of stem cell transformation and application in clinical practice. However, the transcription factor induction system based on viruses has many safety risks such as virus reactivation or instability of the host genome caused by exogenous gene insertion, which cannot be ignored. Although iPS technology has broad application prospects, there are still several important defects that need to be overcome before it can be used clinically: (1) This technology requires virus-mediated gene transfer, and the introduction of viruses can lead to genomic instability, and the integration of viruses has the potential to interfere with normal gene function. (2) More importantly, the genes used in this technology contain clear oncogenes, which greatly increases its possibility of causing cancer and greatly reduces its application value.

[0004] In recent years, stem cell research has achieved many milestones, which has greatly promoted the rapid development of stem cells and regenerative medicine. Somatic cell reprogramming technology makes it very easy for people to obtain pluripotent stem cells and various lineage functional cells, which greatly promotes cell therapy, drug screening, disease model establishment and its mechanism research and application. At present, somatic cell reprogramming can be achieved through nuclear transplantation, cell fusion and viral vector-mediated transcription factor induction, but these somatic cell reprogramming methods have complex operations, potential safety issues such as exogenous gene insertion and viral reactivation, which undoubtedly severely limits the widespread application of somatic cell reprogramming technology in clinical disease treatment. Small molecule compounds have been used as drugs for thousands of years, and their safety has been verified or will undergo strict and safe system testing and evaluation. The mode of action is controllable and reversible, and it is easier to understand and accept emotionally and psychologically. It is of great significance in basic research and clinical application of cell fate transformation. Therefore, the study of small molecule compounds inducing cell reprogramming has gradually become one of the most popular topics in the field of stem cells and regenerative medicine. Small molecule compounds usually refer to biological functional small molecules with a molecular weight of less than 1000 Daltons, mainly including two types: naturally occurring and artificially synthesized. Compared with the above-mentioned induction methods, small molecule compounds have been widely used in somatic cell reprogramming and in vivo transdifferentiation in recent years due to their advantages such as easy synthesis, standardization, controllable and reversible effects, and safety. Therefore, a new safe and controllable reprogramming technology method is urgently needed to be developed. Small molecule compounds are considered to be the most promising new way to induce pluripotent stem cells due to their simplicity, safety, effectiveness and strong operability. Chemical small molecules can interact with specific proteins and achieve the regulation of cell fate by activating or inhibiting the activity of specific target proteins. Small molecules regulate cell fate mainly through the following four mechanisms: ① Regulate related cell signaling pathways. Interact with cell signaling pathway proteins to regulate downstream transcriptional activation by activating or inhibiting signaling pathway-related proteins; ② Regulate cell-related epigenetic states. By interacting with key enzymes or other proteins that regulate the epigenetic state of cells, the epigenetic state of cells is changed; ③ Regulate the metabolic state of cells; ④ Regulate the cytoskeleton. Small molecule compounds can not only promote reprogramming based on exogenously expressed transcription factors, but also completely replace transcription factors to achieve chemical reprogramming completely mediated by chemical small molecules.

[0005] In 2013, Deng Hongkui's team at Peking University reported for the first time in the journal Science that mature somatic cells can be reprogrammed into pluripotent stem cells similar to embryonic stem cells using only a few chemical small molecule combinations. This pioneered the chemical reprogramming technology for cell fate regulation and opened up a new cell fate regulation pathway. Serving human health is one of the ultimate goals of life sciences and regenerative medicine, and it is also one of the ultimate ideals of every scientist. Therefore, based on the successful application of small molecule compounds to induce mouse somatic cell reprogramming, many scientists have continued to explore the use of small molecule compounds to induce human cell reprogramming. However, due to species differences, the effects of small molecule compounds in the mouse system cannot be reproduced in the human system, which indirectly explains why most drugs have good effects in mouse system research but have no obvious therapeutic effects in clinical applications.

[0006] Since there are many difficulties and challenges in the exploration of completely small molecule compounds to reprogram human cells, such as the lack of an efficient reprogramming induction system and the inefficiency and time-consuming molecular barriers to human cell reprogramming, the revelation of the molecular barriers has not yet been clarified. Since 2013, although many domestic and foreign research teams have made a lot of attempts based on the chemical reprogramming of mice, they have not been able to solve the problem of chemical reprogramming of human adult cells. Due to species differences, the experience and understanding of the mouse reprogramming system cannot be reproduced in the human system and there is a lack of an efficient induction factor-mediated human cell reprogramming culture system, so the exploration of completely small molecule compounds to reprogram human cells has encountered many difficulties. Establishing an efficient and determined transcription factor-induced human cell reprogramming system has made it generally believed in the field of stem cell chemistry that the epigenetic restrictions of human adult cells are extremely strict, and it is likely that human adult cells cannot be induced into a pluripotent state through chemical reprogramming. In 2022, Deng Hongkui's team took the lead in reporting in the journal Nature that mature somatic cells can be reprogrammed into pluripotent stem cells similar to embryonic stem cells using only a few chemical small molecule compositions, providing a new paradigm for studying the fate regulation of human somatic cells and the preparation and transformation of stem cells. Compared with the transcription factor overexpression regulation method pioneered by Shinya Yamanaka, chemical reprogramming technology has significant advantages in precisely regulating cell fate. Chemical small molecules can regulate multiple targets and signal pathways simultaneously through a variety of combinations, and orderly and precise cell reprogramming can be achieved by changing the concentration and action time of small molecules. Research on the mechanism of chemical reprogramming further shows that chemical reprogramming is a staged and orderly regulated process. In the process of inducing human hCiPSCs cells, the cells are first induced to an intermediate state similar to the regeneration of lower animal limbs, and then induced to an intermediate state of extraembryonic endoderm, and finally reach and stabilize in a pluripotent state. The chemical reprogramming process presents the characteristics of step-by-step and reverse development, which is similar to the regeneration process of lower animals: somatic cells start to dedifferentiate after responding to exogenous damage signal stimulation, and produce intermediate state cells that mediate tissue regeneration. Chemical reprogramming is a new generation of human iPSCs preparation technology independently developed by my country after "nuclear transplantation" and "transcription factor induction", which has broken through the "bottleneck" problem of underlying technology for the development of stem cells and regenerative medicine in my country. The chemically induced reprogramming method has opened up a new way to achieve somatic cell reprogramming. It is a leap forward in somatic cell reprogramming technology, providing an ideal cell source for future cell therapy and artificial organs. It may even make it possible for humans to directly change the fate of cells in vivo by using small molecule compounds in the future.

[0007] Most of the small molecule compounds currently used are derived from clinical drugs or drugs undergoing clinical trials. They are relatively safe, with controllable and reversible effects, overcoming the above-mentioned safety issues, and are increasingly being used in somatic cell reprogramming research. At present, chemical small molecule reprogramming has been widely used in stem cell and regenerative medicine-related research. Chemical reprogramming can induce astrocytes to be converted into neurons in situ in vivo, induce fibroblasts to be converted into cardiomyocytes, etc., providing a new technical means for in situ regeneration of tissues and organs in vivo. In addition, chemical small molecules can also be used to achieve long-term maintenance of functional cells such as primary liver cells, hematopoietic stem cells and intestinal organoids in vitro. These research advances fully demonstrate the technical advantages of chemical small molecules in precisely regulating cell fate. The discovery of CiPS technology will make these clinical applications safer and more reliable, and may even enable in situ regeneration. In addition, CiPS cells can also be used to manufacture artificial organs in vitro, such as cartilage, brain, kidney, heart, etc., which may provide sources for organ regeneration and organ transplantation in the future. If this goal is achieved, many difficult-to-treat diseases will have new solutions, and the entire field of regenerative medicine will also undergo new changes.

[0008] Compared with traditional methods, the advantages of chemical small molecule reprogramming include simple operation, strong spatiotemporal controllability, reversible effects, and highly controllable cell reprogramming process. In addition, chemical reprogramming technology circumvents the safety issues caused by traditional transgenic operations and is expected to become a safer clinical treatment method. Looking to the future, small molecule compound reprogramming is expected to provide new solutions to major problems such as the regeneration of tissues and organs. By injecting chemical small molecules into the vitreous cavity of mice, the induction of optic nerve regeneration in vivo was initially achieved. Chemical reprogramming can reprogram somatic cells from elderly individuals into hCiPSCs, indicating that the use of chemical small molecules has the possibility of reversing epigenetic marks related to aging. These works show the potential of chemical reprogramming in promoting cell tissue and organ regeneration and fighting aging.

[0009] The chemical reprogramming technology is not only fast, stable and efficient, but more importantly, the chemical composition used in the program is clear, independent of feeder cells and serum. These properties enable the hCiPSCs obtained by chemical reprogramming to better meet the needs of clinical applications, laying the foundation for the establishment of human hCiPSCs cell lines that meet the standards of clinical translational applications, and making it a key step towards clinical application. Compared with the transgenic overexpression transcription factors developed by Yamanaka, chemical reprogramming has technical advantages such as non-integration into the genome, reversible effects, simple and efficient operation, etc. Therefore, hCiPSCs technology is safer, simpler and easier to standardize, and has a broader clinical application prospect. Chemical reprogramming using pure small molecule combinations is an important system for studying cell fate regulation. As an emerging interdisciplinary subject, stem cell chemical biology uses small molecules to precisely regulate cell fate and function in vitro and in vivo, and has broad application prospects in basic research, anti-tumor, anti-aging, regenerative medicine and other fields. Chemical reprogramming is one of the focuses in the field of stem cell chemical biology. In the future, if new technologies such as multi-omics, deep learning and artificial intelligence are combined, it will be able to further accelerate the application of chemical reprogramming technology in the precise regulation of cell fate, providing new hope for solving problems such as the regeneration of tissues and organs and anti-aging.

[0010] The most convenient sources for obtaining starting cells for human cell reprogramming mainly include: skin, blood, urine, amniotic fluid, placenta, and umbilical cord tissues, which have different reprogramming potentials and efficiencies. Compared with cells from other human tissues, umbilical cord tissue cells have many advantages such as non-invasiveness, less exposure to in vitro radiation, convenient sample collection, and simple separation and acquisition operations, which can be achieved in a simple laboratory environment and facilitate future promotion and use. In addition, the use of human umbilical cord tissue-derived cells for reprogramming has the following advantages, which can better meet the needs of future clinical treatment: ① Umbilical cord tissue is rich in sources, easy to obtain, and has no adverse effects such as invasive damage to donors; ② Umbilical cord tissue is clinically medical waste, and most of it is discarded for free on a daily basis, which will not cause ethical and legal constraints; ③ Umbilical cord tissue-derived cells have great differentiation potential and strong proliferation ability, and can be amplified in large quantities in vitro to quickly meet the requirements of clinical use; ④ It is easy to carry out large-scale industrial preparation; ⑤ It is safe and has no risk of viral infection and other advantages. These advantages undoubtedly make umbilical cord tissue-derived cells a type of cell that can be reprogrammed and has high application potential in the field of cell therapy, making it an ideal seed cell in stem cell therapy research. Technical platforms such as complete small molecule compound reprogramming, animal-free induction culture system and GMP-grade cell production workshop will enable functional cells derived from human umbilical cord tissue to be promoted to clinical applications faster and safer. The use of small molecule compounds to regulate cell fate has the advantages of simple operation, easy synthesis and structural adjustment, rapid biological effects, easy to accurately adjust biological effects by adjusting concentration, and avoidance of exogenous gene manipulation. Summary of the invention

[0011] The technical problems to be solved by the present invention are:

[0012] Provide a serum-free culture method for inducing the conversion of hUC-MSCs into hiPSCs by small molecule compounds. The purpose is to simply, quickly and efficiently reprogram hUC-MSCs through several small molecule compounds to obtain hiPSCs cells with long-term passage.

[0013] The technical solution of the present invention to solve the above technical problems is as follows:

[0014] A serum-free culture method for inducing hUC-MSCs to transform into hiPSCs by small molecule compounds, wherein the culture system includes a one-stage culture system, a two-stage culture system, a three-stage culture system and a four-stage culture system;

[0015] The first stage culture system comprises: KnockOut DMEM (Gibco, 10829018), knockout serum replacement (KSR) (Gibco, 10828028), N2 additive (Gibco, 17502-048), B27 additive (Gibco, 17504-044), GlutaMax, NEAA, mM 2-mercaptoethanol, L-ascorbic acid 2-phosphate (Vc 2P) (Sigma-Aldrich, A8960), LiCl (Sigma-Aldrich, L4408), nicotinamide (NAM) (Sigma-Aldrich, 72340), AlbuMax-II (Gibco, 11021045), bFGF, Vc, R406, GSK3β inhibitor, TGFβR inhibitor, RAR activator, VEGFR / PDGFR inhibitor;

[0016] The second stage culture system includes: KnockOut DMEM, KSR, N2 additive (Gibco, 17502-048), B27 additive (Gibco, 17504-044), GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, LiCl, NAM, bFGF (Origene, TP750002), Vc, R406, GSK3β inhibitor, TGFβR inhibitor, RAR activator, C-jun N-terminal kinase inhibitor, MAO inhibitor, Smoothened receptor agonist, DNA methyltransferase inhibitor;

[0017] The three-stage culture system includes KnockOut DMEM, KSR, N2 additive (Gibco, 17502-048), B27 additive (Gibco, 17504-044), GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, AlbuMax-II, recombinant human heregulin β-1 (HRG) (PeproTech, 100-03), bFGF, Vc, R406, GSK3β inhibitor, TGFβR inhibitor, ROCK inhibitor, MEK1 / 2 inhibitor, MAO inhibitor, DOT1L histone methyltransferase inhibitor, and HDAC inhibitor;

[0018] The four-stage culture system includes KnockOut DMEM, KSR, N2 additive (Gibco, 17502-048), B27 additive (Gibco, 17504-044), GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, recombinant human heregulin β-1 (HRG) (PeproTech, 100-03), bFGF, Vc, R406, GSK3β inhibitor, MEK1 / 2 inhibitor, ROCK inhibitor, and HDAC inhibitor.

[0019] The beneficial effects of the present invention are:

[0020] The present invention can use the above-mentioned culture system to simply, quickly and efficiently use small molecule compounds to induce hUC-MSCs into hiPSCs in a serum-free culture system, and obtain iPSC cells with long-term passage, which provides a promising way for regenerative medicine applications. Complete small molecule compound reprogramming, animal-free induction culture system and GMP-level cell production workshop and other technical platforms will enable functional cells derived from human umbilical cord tissue to be more quickly and safely promoted to clinical applications. The use of small molecule compounds to regulate cell fate has the advantages of simple operation, easy synthesis and structural adjustment, rapid biological effects, easy to accurately adjust biological effects by adjusting concentrations, and avoidance of exogenous gene manipulation. Small molecule compound reprogramming technology circumvents the safety issues caused by traditional transgenic operations and is expected to become a safer clinical treatment method. Looking to the future, small molecule compound reprogramming is expected to provide new solutions to major problems such as achieving tissue and organ regeneration. In addition, CiPS cells can also be used to manufacture artificial organs in vitro, such as cartilage, brain, kidney, heart, etc., which may provide sources for organ regeneration and organ transplantation in the future. If this goal is achieved, many difficult-to-treat diseases will be given new solutions, and the entire field of regenerative medicine will also undergo new changes.

[0021] The serum-free culture method for inducing hUC-MSCs to transform into hiPSCs by using small molecule compounds is characterized by:

[0022] The GSK3β inhibitors include CHIR999021 and SB216763;

[0023] The TGFβR inhibitors include SB-431542, 616452, and GW788388;

[0024] The RAR activators include TTNPB and Tamibarotene;

[0025] The VEGFR / PDGFR inhibitors include AL39324 and RG3635;

[0026] The ROCK inhibitors include Y-27632 and BAY-549;

[0027] The C-jun N-terminal kinase inhibitors include SP600125 and JNK-IN-7;

[0028] The MAO inhibitors include iproniazid;

[0029] The MEK1 / 2 inhibitors include PD 184352 and Trametinib;

[0030] The HDAC inhibitor includes VPA;

[0031] The DOT 1L histone methyltransferase inhibitors include DZN ep and GSK343;

[0032] The B-Raf inhibitor includes at least one of SB590885 and GDC 0879.

[0033] The Smoothened receptor agonists include SAG HCl and Sonic Hedgehog;

[0034] The serum-free culture method for inducing hUC-MSCs to transform into hiPSCs by using small molecule compounds is characterized by:

[0035] The amounts of the components in the first stage culture system are as follows: KnockOut DMEM (Gibco, 10829018), 15% knockout serum replacement (KSR) (Gibco, 10828028), 2% N2 supplement (Gibco, 17502-048), 3% B27 supplement (Gibco, 17504-044), 15% FBS, 2% GlutaMax, 2% NEAA, 0.1 mM 2-mercaptoethanol, 50 μg / ml L-ascorbic acid 2-phosphate (Vc 2P) (Sigma-Aldrich, A8960), 5 mM LiCl (Sigma-Aldrich, L4408), 1 mM nicotinamide (NAM) (Sigma-Aldrich, 72340), 2 mg / ml AlbuMax-II (Gibco, 11021045), 25 ng / ml bFGF, 50 μg / ml Vc, 1 μM R406, 30 μM TGFβR inhibitor, 35 μM GSK3β inhibitor, 6 μM RAR activator, 3 μM VEGFR / PDGFR inhibitor;

[0036] The dosage of each component in the two-stage culture system is as follows: KnockOut DMEM, 15% KSR, 2% N2 additive (Gibco, 17502-048), 3% B27 additive (Gibco, 17504-044), 2% GlutaMax, 2% NEAA, 0.1mM 2-mercaptoethanol, 50μg / ml Vc2p, 5mM LiCl, 2mMNAM, 50ng / ml bFGF (Origene, TP750002), 50μg / ml Vc, 1μM R406, 40μM GSK3β inhibitor, 35μM TGFβR inhibitor, 6μM RAR activator, 3μM C-jun N-terminal kinase inhibitor, 35μM MAO inhibitor, 1.5μM Smoothened receptor agonist, 30μM DNA methyltransferase inhibitor;

[0037] The dosage of each component in the three-stage culture system is as follows: KnockOut DMEM, 15% KSR, 2% N2 additive (Gibco, 17502-048), 3% B27 additive (Gibco, 17504-044), 3% GlutaMax, 2% NEAA, 0.1 mM 2-mercaptoethanol, 50 μg / ml Vc2p, 5 mg / ml AlbuMax-II, 20 ng / ml recombinant human heregulin β-1 (HRG) (PeproTech, 100-03), 25 ng / ml bFGF, 50 μg / ml Vc, 1 μM R406, 3 μM GSK3β inhibitor, 30 μM TGFβR inhibitor, 35 μM ROCK inhibitor, 3 μM MEK1 / 2 inhibitor, 30 μM MAO inhibitor, 0.6 μM DOT1L histone methyltransferase inhibitor, 1500 μM HDAC inhibitor;

[0038] The dosage of each component in the four-stage culture system is as follows: KnockOut DMEM, 15% KSR, 2% N2 additive (Gibco, 17502-048), 3% B27 additive (Gibco, 17504-044), 3% GlutaMax, 2% NEAA, 0.1mM 2-mercaptoethanol, 50μg / ml Vc2p, 20ng / ml recombinant human heregulinβ-1 (HRG) (PeproTech, 100-03), 25ng / ml bFGF, 50μg / ml Vc, 1μM R406, 3μM GSK3β inhibitor, 3μM MEK1 / 2 inhibitor 1500μM HDAC inhibitor, 30μM ROCK inhibitor.

[0039] The serum-free culture method for inducing hUC-MSCs to transform into hiPSCs by small molecule compounds includes the following four specific stages:

[0040] ① Forming epithelial cells: using the above-mentioned first-stage culture system to culture hUC-MSCs and induce them into epithelial cells;

[0041] ② Forming multilayer cell colonies: culturing the epithelial cells using the two-stage culture system to induce them into multilayer cell colonies;

[0042] ③ Expanding the multilayer cell colony: using the three-stage culture system to expand and culture the multilayer cell colony to induce it into an expanded multilayer cell colony;

[0043] ④ Forming induced pluripotent stem cells: The amplified multilayer cell colonies are cultured using the four-stage culture system to induce them into induced pluripotent stem cells.

[0044] (7) The beneficial effects of adopting the above further scheme are: using human umbilical cord tissue-derived cells for reprogramming has the following advantages, which can better meet the needs of future clinical treatment: ① Umbilical cord tissue is rich in sources, easy to obtain, and has no adverse effects such as invasive damage to donors; ② Umbilical cord tissue is clinically medical waste, and most of it is discarded for free on a daily basis, which will not cause ethical and legal constraints; ③ Umbilical cord tissue-derived cells have great differentiation potential and strong proliferation ability, and can be amplified in large quantities in vitro to quickly meet the requirements of clinical use quantity; ④ It is easy to carry out large-scale industrial preparation; ⑤ It is safe and has no risk of viral infection and other advantages. These advantages undoubtedly make umbilical cord tissue-derived cells a type of cell with high application potential in the field of cell therapy for reprogramming, making it an ideal seed cell in stem cell therapy research. Technical platforms such as complete small molecule compound reprogramming, animal-free induction culture system and GMP-grade cell production workshop will enable human umbilical cord tissue-derived functional cells to be promoted to clinical application faster and safer. DETAILED DESCRIPTION

[0045] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased through regular channels.

[0046] The immunofluorescence staining and immunohistochemical staining used in the following examples are described as follows:

[0047] 1. Immunofluorescence identification of hCiPSCs

[0048] Immunofluorescence staining includes the following steps:

[0049] a) When the cells grow to 60-70%, the experiment begins;

[0050] b) Aspirate the culture medium and wash with PBS 2-3 times;

[0051] c) Fix with 4% paraformaldehyde at room temperature for 20 minutes; wash with PBS 2-3 times, 3-5 minutes each time;

[0052] d) 0.1% Triton X 100 (dissolved in PBS), add 150 μL to each well of a 24-well plate, permeabilize the membrane for 10 min, and wash 2-3 times with PBS, 3-5 min / time;

[0053] e) 2% BSA (dissolved in PBS), blocked at room temperature for 1-2 hours

[0054] f) Primary antibody incubation: dilute the primary antibody (use 1% BSA for the ratio as per the instructions), take 50 μL of the primary antibody dilution and drop it onto a small piece of sealing film, then cover the cell surface of the slide onto the primary antibody droplet, and incubate at 4°C overnight in a dark place.

[0055] g) Rewarm at room temperature for 30 min, and wash 2-3 times with PBS, 3-5 min / time;

[0056] h) Secondary antibody incubation: dilute the secondary antibody (refer to the instructions for the ratio), take 50 μL of the secondary antibody dilution solution and add it to a small piece of sealing film, then cover the cell surface of the glass slide on the secondary antibody droplet, at room temperature, away from light, for 1-2 hours

[0057] i) Wash with PBS 2-3 times, 3-5 min / time, and protect from light;

[0058] j) Dilute DAPI at 1:5000 and add DAPI staining solution at room temperature in the dark for 3-5 minutes

[0059] k) Wash with PBS 2-3 times, and wash thoroughly on a shaker;

[0060] l) Sealing: Add a drop of anti-fading sealing agent, put on a coverslip, observe the corresponding fluorescence results under a laser confocal microscope and take pictures.

[0061] m) Storage: Store at 4°C in a humidified box.

[0062] 2. Isolation, culture and identification of hUC-MSCs

[0063] 2.1 Isolation and culture of hUC-MSCs

[0064] a) Take the umbilical cords of healthy fetuses delivered by cesarean section at full term, soak them in sterile saline containing 1% penicillin and streptomycin, and store them on ice;

[0065] b) Take out the newborn's umbilical cord from the sterile saline solution containing 1% double antibody, and rinse it with saline several times until the blood stains are washed away. Discard the deformed part of the blood vessel and the two ends of the opening;

[0066] c) Cut the umbilical cord into 3-4 cm pieces, use tissue forceps to remove the umbilical cord skin and blood vessels (2 arteries and 1 vein, cut along the vein, then carefully tear out the venous lining, and directly pull out the artery with force), leaving the lining, which is Whalton's Jelly, and wash it in saline for 2-3 times;

[0067] d) Transfer the umbilical cord to a 1.5 mL centrifuge tube, add a 1:1 mixture of 0.1% collagenase type 4 and pancreatin, cut it into pieces, and digest for 1 to 2 hours depending on the situation;

[0068] e) Add 3-5 times the volume of complete medium containing 10% serum to terminate the action of trypsin. Filter with a cell sieve, centrifuge the filtrate containing cells at 300g for 5 minutes, and remove the supernatant;

[0069] f) The cells were seeded in a 10 cm culture dish containing 10% serum DMEM / F12 medium. The culture medium was replaced after three or four days of culture to remove non-adherent cells. The medium was then replaced every 3 days until the cells were fully confluent and passaged. At this time, the cells were recorded as P0.

[0070] 2.2 Recovery of hUC-MSCs

[0071] Before thawing cells, preheat a 37°C water bath and preheat DMEM / F12 medium. Take the cells out of the liquid nitrogen tank, quickly place them in a 37°C water bath and shake them to melt quickly until only a small ice crystal remains. Take them out of the water bath, spray them with alcohol and wipe them dry before transferring them to a biosafety cabinet. Mix the cell suspension and transfer it to a 15mL centrifuge tube containing DMEM / F12 medium, add dropwise, centrifuge for 5 minutes, remove the supernatant, resuspend and count with complete medium, inoculate them in a cell culture dish of appropriate size, shake well, and place them in a cell culture incubator.

[0072] 2.3 Passaging of hUC-MSCs

[0073] When the cells grow to 80-90% fusion rate, discard the culture medium, wash twice with PBS, remove the excess culture medium, add appropriate amount of 0.05% trypsin and digest at 37℃ for 2min. Add twice the volume of complete culture medium containing serum to terminate digestion, blow the cells several times to accelerate the cells from the surface of the culture dish, and collect them in a centrifuge tube for cell counting. Centrifuge the cell suspension at 300g for 5min and remove the supernatant. Subculture and amplify at a cell density of 5000 cell cm-2.

[0074] 2.4 Cryopreservation of hUC-MSCs

[0075] When the cells grow to 80-90% fusion rate, cryopreserve the cells and keep them as seeds: discard the culture medium, wash twice with PBS, add appropriate amount of trypsin to digest for 2 minutes, 37°C, terminate digestion with twice the volume of complete culture medium, blow the cells to collect them in a 15mL centrifuge tube, centrifuge, and remove the supernatant. Resuspend the cell pellet with complete cell culture medium, count the cells, adjust the density to 2×106cells mL-1, add the same volume of 2× freezing solution to the cell suspension in the centrifuge tube, mix while adding, and then transfer to the cryopreservation tube, mark the cell type, generation, operator, time and cell amount and other information. After placing the cryopreservation tube in a program freezing box overnight, transfer it to liquid nitrogen for long-term storage.

[0076] 2.1 Identification of hUC-MSCs

[0077] 2.1.1 Flow cytometry of hUC-MSCs

[0078] After digestion and collection of cells, wash them twice again with 5mL PBS, then add 100μL PBS per 1×106 cells to resuspend the cell pellet at the bottom of the tube, distribute it into multiple EP tubes, add the corresponding antibodies, and leave one tube without antibody as a control. After incubation in dark for 30min with 4mL, wash twice with PBS, and finally resuspend the cells in each tube with 500μL PBS, analyze and plot on a flow cytometer using FlowJo_V10 to identify MSCs surface markers.

[0079] 2.1.2 Identification of adipogenic differentiation of hUC-MSCs

[0080] hUC-MSCs were cultured at 2×10 4 cm -2 The cells were seeded at a density of 1.50 μg / mL in a 24-well plate, and 15% FBS and 1 ng mL -1 0.5 mL of low-glucose DMEM culture medium containing bFGF was added; when the cells reached 80% confluence, the induction culture medium (high-glucose DMEM, 10% FBS, hydrocortisone, isobutylmethylxanthine, indomethacin) was changed, and the medium was changed every 3 to 4 days; Oil red O staining was performed for identification after 2 weeks of induction.

[0081] 2.1.3 Identification of osteogenic differentiation of hUC-MSCs

[0082] hUC-MSCs were cultured at 2×10 4 cm -2 The cells were seeded at a density of 100 μg / mL in a 24-well plate and cross-mixed. After 24 h of cell attachment, each well was replaced with 0.5 mL of osteogenic induction solution (high-glucose DMEM, 10% FBS, sodium β-glycerophosphate, dexamethasone, ascorbic acid). The solution was changed every 2-3 days and the induction was observed under a microscope. At 21 days, alizarin red staining was performed, the culture medium was aspirated, the cells were washed 3 times with 1xPBS, fixed with 95% ethanol for 10 min, rinsed 3 times with double distilled water, 200 μL of 2% alizarin red dye (pH=4.2) was added, and the cells were stained at room temperature for 30 min. The excess dye was washed off with double distilled water and observed under a microscope.

[0083] 2.1.4 Identification of chondrogenic differentiation of hUC-MSCs

[0084] Induction medium: high-glucose DMEM + 1% penicillin-streptomycin solution + sodium pyruvate + ascorbic acid + dexamethasone + 1% ITS + TGF-β3.

[0085] Cell culture: Take 2x10 5 The cell suspension was dispensed into 15 mL centrifuge tubes, centrifuged again and the supernatant was carefully removed. The chondrogenic induction solution was gently added along the tube wall and placed in a CO2 incubator. The tube cap was first tightened and then loosened half a turn. The solution was changed every 3 days and the induction condition was observed. Alcian blue staining was performed on day 21. The culture medium was discarded, the tubes were washed 3 times with 1×PBS, fixed with 4% paraformaldehyde for 30 min, washed 3 times with 1×PBS, stained with Alcian blue dye overnight (200 μL / tube), washed 3 times with 1×PBS until it did not fade, and pictures were taken under a stereomicroscope.

[0086] 3. Specific implementation of inducing hUC-MSCs into hiPSCs

[0087] 3.1 The first stage of induction of hUC-MSCs (12 days):

[0088] (1) taking out hUC-MSCs target cells from the incubator, removing the original culture medium, adding the first culture medium, adding 1 mL of culture medium to each well of the 12-well plate, mixing, and placing in a 37° C. carbon dioxide incubator;

[0089] (2) The medium was changed every 4 days, and cell deformation was continuously observed during the process. Usually, a single layer of epithelial-like cells began to appear after about 4 days of induction. The induction was continued until the confluence of the single layer of epithelial cells approached 100%, and the first stage of induction was terminated.

[0090] 3.2 Second stage induction (12 days).

[0091] (1) Take out the cells that have completed the first stage of induction from the incubator, remove the original culture medium, add the second stage culture medium, add 1 mL of culture medium to each well of the 12-well plate, mix well, and place in a 37°C carbon dioxide incubator;

[0092] (2) The medium is changed every 4 days. Usually, after about 4 days of induction, multilayer cell clones will appear. Continue culturing until a large number of multilayer clones are produced, ending the second stage of induction.

[0093] 3.3 The third stage of induction (8 days):

[0094] (1) Take out the cells after the second stage of induction from the incubator, remove the original culture medium, add the three stages of culture medium, add 1 mL of culture medium to each well of the 12-well plate, mix well, and place in a 37°C carbon dioxide incubator; change the medium every 4 days.

[0095] 3.4 The fourth stage of induction (6-8 days):

[0096] (1) Take out the cells after the second stage of induction from the incubator, remove the original culture medium, add the four stages of culture medium, add 1 mL of culture medium to each well of the 12-well plate, mix well, and place in a 37°C carbon dioxide incubator; change the medium every 4 days.

[0097] (Generally, smooth raised pluripotent stem cell clones can be observed in about 6-8 days).

[0098] 3.5 Single cell line establishment

[0099] 1) Prepare the matrix-coated culture plates: This process needs to be completed one day in advance.

[0100] (1) Prepare Laminin-521 coated plates according to the instructions;

[0101] (2) Thaw a 1 mL vial of Laminin-521 (100 μg) at 4°C;

[0102] (3) Dilute Laminin-521 with (containing calcium and magnesium) at a ratio of 1:40, i.e., add 1 mL of Laminin-521 to 39 mL of DPBS. After mixing, add 700 μL to each well of a 12-well plate (add the same volume to other wells, and the final mass per well is 1.75 μg);

[0103] (4) Incubate overnight in a 37°C CO2 incubator. After encapsulation, use directly or seal and store at 4°C (no more than 1 week at 4°C).

[0104] 2) Single cell replating culture:

[0105] (1) Take out the cells from the incubator, observe them under a microscope, select wells with good status and relatively more clones, add 0.5 mL / well of basal culture medium DF12, and wash once;

[0106] (2) Add 0.5 mL of 37°C preheated digestive enzyme Accutase to each well of the 12-well plate and place in a 37°C CO2 incubator for 5-8 minutes;

[0107] (3) Add an equal volume of DF12 and pipette repeatedly until no lumps are evident;

[0108] (4) After collecting the cell suspension, centrifuge at 400g for 5 minutes;

[0109] (5) Carefully remove the supernatant, resuspend the cells in culture medium F, and inoculate them onto the Laminin521-coated plate;

[0110] (6) Depending on the number of cells and clones, the inoculation density is usually 1:6-1:12;

[0111] (7) Shake well and place in a 37°C CO2 incubator for culture. Change the medium every three days. Obvious clones can be observed in about 3-6 days.

[0112] (8) Usually after 6 days, when the clones are slightly larger, you can switch to mTESR TM Continue culturing in Plus medium for 6 days, changing the medium every 2 days. When the clones grow to a relatively large size, usually with a diameter greater than 80 μm, you can proceed to the next step.

[0113] 3) Select a single clone to establish a cell line:

[0114] (1) Take out the cells from the incubator, observe the clones under a microscope, and mark the single clones to be picked with a marker pen. The selection criteria are: full morphology, dense clones, clear edges, undifferentiated homogeneous clones, and a size of 80-150 μm. Try to choose clones that are far apart for easy operation;

[0115] (2) Place the stereoscope in a biological safety cabinet in advance and sterilize it under ultraviolet light for 30 minutes;

[0116] (3) Preparation of inoculation medium: mTESR TM Plus was added with a final concentration of 10 μ m Y27632;

[0117] (4) Aspirate the original culture medium, add 0.5 mL DF12, and wash once to remove residual dead cells;

[0118] (5) Add 0.3 mL / well (12-well plate) of digestion solution ReleSR, shake well, and digest at room temperature for 2 minutes; aspirate the digestion solution and digest at 37°C for 2-3 minutes;

[0119] (6) Under a stereomicroscope, carefully scrape off the colonies with a small gun tip or glass needle, absorb them with 20 μL of inoculation medium, and disperse them into small colonies;

[0120] (7) Aspirate the coating solution from the matrix-coated culture plate and add inoculation culture solution at a volume of 0.5 mL per 1 / 24 well;

[0121] (8) Inoculate a small piece of each monoclonal clone into a matrix-coated culture plate, usually one monoclonal clone is inoculated into 1 / 24 well;

[0122] (9) Usually 10-20 single clones are picked, but it is possible that the final number of clones is less than 10 clones;

[0123] (10) After picking, all remaining clones are resuspended in inoculation medium, inoculated onto coated plates, and mixed cultured for further cloning;

[0124] (11) Shake well and place in an incubator;

[0125] (12) On the second day, observe the adhesion of the clones. Usually, almost all clones can adhere well to the wall.

[0126] (13) Change the solution, remove the liquid, and add fresh mTESR TM Plus culture medium;

[0127] (14) Depending on the growth rate and status of the cells, change the medium every 1-2 days until the clones grow large enough for subculture.

[0128] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are merely exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 This is a diagram of cell morphology changes in Example 1 of the present invention;

[0130] Figure 2 The iPSC cells of Example 1 of the present invention have the ability to differentiate into three germ layers.

Claims

1. A serum-free culture method for inducing hUC-MSCs to transform into hiPSCs using small molecule compounds, characterized in that: The culture system includes four stages: a one-stage culture system, a two-stage culture system, a three-stage culture system and a four-stage culture system; The first stage culture system comprises: KnockOut DMEM (Gibco, 10829018), knockout serum replacement (KSR) (Gibco, 10828028), N2 additive (Gibco, 17502-048), B27 additive (Gibco, 17504-044), GlutaMax, NEAA, mM 2-mercaptoethanol, L-ascorbic acid 2-phosphate (Vc 2P) (Sigma-Aldrich, A8960), LiCl (Sigma-Aldrich, L4408), nicotinamide (NAM) (Sigma-Aldrich, 72340), AlbuMax-II (Gibco, 11021045), bFGF, Vc, R406, GSK3β inhibitor, TGFβR inhibitor, RAR activator, VEGFR / PDGFR inhibitor; The second stage culture system includes: KnockOut DMEM, KSR, N2 additive (Gibco, 17502-048), B27 additive (Gibco, 17504-044), GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, LiCl, NAM, bFGF (Origene, TP750002), Vc, R406, GSK3β inhibitor, TGFβR inhibitor, RAR activator, C-jun N-terminal kinase inhibitor, MAO inhibitor, Smoothened receptor agonist, DNA methyltransferase inhibitor; The three-stage culture system includes KnockOut DMEM, KSR, N2 additive (Gibco, 17502-048), B27 additive (Gibco, 17504-044), GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, AlbuMax-II, recombinant human heregulin β-1 (HRG) (PeproTech, 100-03), bFGF, Vc, R406, GSK3β inhibitor, TGFβR inhibitor, ROCK inhibitor, MEK1 / 2 inhibitor, MAO inhibitor, DOT1L histone methyltransferase inhibitor, and HDAC inhibitor; The four-stage culture system includes KnockOut DMEM, KSR, N2 additive (Gibco, 17502-048), B27 additive (Gibco, 17504-044), GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, recombinant human heregulin β-1 (HRG) (PeproTech, 100-03), bFGF, Vc, R406, GSK3β inhibitor, MEK1 / 2 inhibitor, ROCK inhibitor, and HDAC inhibitor.

2. The serum-free culture method for inducing hUC-MSCs to transform into hiPSCs by small molecule compounds according to claim 1, characterized in that: The GSK3β inhibitors include CHIR999021 and SB216763; The TGFβR inhibitors include SB-431542, 616452, and GW788388; The RAR activators include TTNPB and Tamibarotene; The VEGFR / PDGFR inhibitors include AL39324 and RG3635; The ROCK inhibitors include Y-27632 and BAY-549; The C-jun N-terminal kinase inhibitors include SP600125 and JNK-IN-7; The MAO inhibitors include iproniazid; The MEK1 / 2 inhibitors include PD 184352 and Trametinib; The HDAC inhibitor includes VPA; The DOT 1L histone methyltransferase inhibitors include DZ Ne p and GSK343; The B-Raf inhibitor includes at least one of SB590885 and GDC 0879. The Smoothened receptor agonists include SAG HCl and Sonic Hedgehog.

3. The serum-free culture method for inducing hUC-MSCs to transform into hiPSCs by small molecule compounds according to claim 1, characterized in that: The amounts of the components in the first stage culture system are as follows: KnockOut DMEM (Gibco, 10829018), 15% knockout serum replacement (KSR) (Gibco, 10828028), 2% N2 supplement (Gibco, 17502-048), 3% B27 supplement (Gibco, 17504-044), 15% FBS, 2% GlutaMax, 2% NEAA, 0.1 mM 2-mercaptoethanol, 50 μg / ml L-ascorbic acid 2-phosphate (Vc 2P) (Sigma-Aldrich, A8960), 5 mM LiCl (Sigma-Aldrich, L4408), 1 mM nicotinamide (NAM) (Sigma-Aldrich, 72340), 2 mg / ml AlbuMax-II (Gibco, 11021045), 25 ng / ml bFGF, 50 μg / ml Vc, 1 μM R406, 30 μM TGFβR inhibitor, 35 μM GSK3β inhibitor, 6 μM RAR activator, 3 μM VEGFR / PDGFR inhibitor; The dosage of each component in the two-stage culture system is as follows: KnockOut DMEM, 15% KSR, 2% N2 additive (Gibco, 17502-048), 3% B27 additive (Gibco, 17504-044), 2% GlutaMax, 2% NEAA, 0.1mM 2-mercaptoethanol, 50μg / ml Vc2p, 5mM LiCl, 2mM NAM, 50ng / ml bFGF (Origene, TP750002), 50μg / ml Vc, 1μM R406, 40μM GSK3β inhibitor, 35μM TGFβR inhibitor, 6μM RAR activator, 3μM C-jun N-terminal kinase inhibitor, 35μM MAO inhibitor, 1.5μM Smoothened receptor agonist, 30μM DNA methyltransferase inhibitor; The dosage of each component in the three-stage culture system is as follows: KnockOut DMEM, 15% KSR, 2% N2 additive (Gibco, 17502-048), 3% B27 additive (Gibco, 17504-044), 3% GlutaMax, 2% NEAA, 0.1 mM 2-mercaptoethanol, 50 μg / ml Vc2p, 5 mg / ml AlbuMax-II, 20 ng / ml recombinant human heregulin β-1 (HRG) (PeproTech, 100-03), 25 ng / ml bFGF, 50 μg / ml Vc, 1 μM R406, 3 μM GSK3β inhibitor, 30 μM TGFβR inhibitor, 35 μM ROCK inhibitor, 3 μM MEK1 / 2 inhibitor, 30 μM MAO inhibitor, 0.6 μM DOT1L histone methyltransferase inhibitor, 1500 μM HDAC inhibitor; The dosage of each component in the four-stage culture system is as follows: KnockOut DMEM, 15% KSR, 2% N2 additive (Gibco, 17502-048), 3% B27 additive (Gibco, 17504-044), 3% GlutaMax, 2% NEAA, 0.1mM 2-mercaptoethanol, 50μg / ml Vc2p, 20ng / ml recombinant human heregulinβ-1 (HRG) (PeproTech, 100-03), 25ng / ml bFGF, 50μg / ml Vc, 1μM R406, 3μM GSK3β inhibitor, 3μM MEK1 / 2 inhibitor, 1500μM HDAC inhibitor, and 30μM ROCK inhibitor.

4. A serum-free culture method for inducing hUC-MSCs to transform into hiPSCs using small molecule compounds, characterized in that: The culture system comprises the one-stage culture system to the four-stage culture system as described in any one of claims 1 to 3.

5. A serum-free culture method for inducing hUC-MSCs to transform into hiPSCs using small molecule compounds, characterized in that: The method comprises the following steps: using the one-stage culture system to the four-stage culture system described in any one of claims 1 to 3 to culture hUC-MSCs to obtain induced pluripotent stem cells.

6. The serum-free culture method for inducing hUC-MSCs to transform into hiPSCs by small molecule compounds according to claim 5, characterized in that: It includes the following 4 specific induction reprogramming stages: ① Forming epithelial cells: using the above-mentioned first-stage culture system to culture hUC-MSCs and induce them into epithelial cells; ② Forming multilayer cell colonies: culturing the epithelial-like cells using the two-stage culture system to induce them into multilayer cell colonies; ③ Expanding the multilayer cell colony: using the three-stage culture system to expand and culture the multilayer cell colony to induce it into an expanded multilayer cell colony; ④ Forming induced pluripotent stem cells: The amplified multilayer cell colonies are cultured using the four-stage culture system to induce them into induced pluripotent stem cells.

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