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Author: Shen Hongfen 1, 3) ** PVN 1) ** Xiao Gaofang 1) Jia Jun 1 double) Xiao Dong 1, 2) *** Kai-Tai Yao 1, 3) *** (1) Cancer Research Institute, Southern Medical University, Guangzhou 510515, China; 2) Institute of Comparative Medicine and Laboratory Animal Center, Southern Medical University, Guangzhou 510515; 3) Institute of Cancer Research, Central South University, Changsha 410078)
Abstract Mainly from the development process of iPS cells, several key steps to obtain iPS cells (such as gene transfer methods, combination of factors required to induce iPS cells, use of small molecule compounds, and somatic cell type selection, etc.), patient or disease-specific iPS cells, In vitro and in vivo induction of differentiation of iPS cells and the clinical application of their derivatives and the feasibility and prospects of preparing genetic modification-free iPS cells are reviewed on the latest research progress of iPS cells. The Japanese and American research groups used four genes to reprogram mouse (August 2006) and human (November to December 2007) somatic cells in vitro into induced pluripotent stem cells (iPS cells). ), in just over two years, the research and attention of iPS cells has exploded. A series of hot issues such as somatic cell reprogramming, dedifferentiation and the source of pluripotent stem cells have once again become the focus and focus of research on stem cells and developmental biology. Like embryonic stem cells (ES cells), iPS cells can differentiate into all cells derived from three germ layers in the body, and then participate in the formation of all tissues and organs of the body. So far, iPS cells have been directed to induce and differentiate into a variety of functional mature cells in vitro. Therefore, iPS cell research not only has important theoretical significance, but also has great application value in regenerative medicine, tissue engineering, and drug discovery and evaluation.
Key words somatic cell reprogramming, embryonic stem cells, induced pluripotent stem cells, differentiation, cell therapy, reprogramming methods without genetic modification, small molecule subject classification number Q813, Q75 DOI: 10.3724/SP.J.1206.2008.00794
Because human embryonic stem cells (ES cells) have great application value in the fields of regenerative medicine, tissue engineering, and drug discovery and evaluation, scientists have tried different ways to achieve somatic cell reprogramming to obtain ES cells or ES cells. Such cells or induced pluripotent stem cells (iPS cells), these pathways mainly include: a. Somatic cell nuclear transfer, b. Reprogramming after fusion of somatic cells and pluripotent cells, c. Incubating differentiated somatic cells in extracts of egg cells or pluripotent stem cells to achieve somatic cell reprogramming, d. Somatic cells are induced to reprogram into iPS cells by specific factors [1~4]. Although pluripotent stem cells can be obtained by using the first three methods, the wide application of these methods has many limitations in terms of technology, cell sources, immune rejection, ethics, religion, and law. However, iPS cells are not restricted by these problems and are simple to prepare Easy to do, so once iPS cells came out, they caused a sensation in the field of life sciences, and were hailed as a new milestone in the field of life sciences. What are iPS cells? Simply put, it is to use gene transfer technology to introduce certain specific factors into animal or human somatic cells, and at the same time, you can selectively add specific small molecules in the culture medium to reprogram somatic cells into pluripotent stem cells. In clonal morphology, growth characteristics, surface markers, gene expression patterns, epigenetic characteristics, embryoid bodies (EBs) formation, teratoma (teratoma) formation and chimeras formation (for Mouse) and other aspects are very similar to ES cells. This article mainly focuses on the development process of iPS cells, several key steps to obtain iPS cells (such as gene transfer methods, combination of factors required to induce iPS cells, use of small molecule compounds, and somatic cell type selection, etc.), patient or disease-specific iPS cells, In vitro and in vivo induction of differentiation of iPS cells, the clinical application of their derivatives, the feasibility and prospects of preparing iPS cells without genetic modification, etc. The latest research progress of iPS cells is reviewed. In addition, for the early and other developments of iPS cells, please refer to other Chinese reviews [1, 5～10]. 1 The development process of iPS cells is shown in Table 1 [11～47]. In August 2006, the Yamanaka team introduced 24 transcription factor permutations and combinations into mouse fibroblasts, and finally determined that there are at least 4 transcription factor combinations—Oct4 , Sox2, c-Myc and Klf4 can reprogram fibroblasts into iPS cells[11]. From November to December 2007, Yamanaka and Thomson groups reprogrammed human somatic cells into iPS cells successively [12, 13] . Since then, the research and attention of iPS cells has exploded (Table 1), and some breakthroughs have been made, such as the establishment of disease-specific human iPS cells [28, 29], and transposon-mediated transgenic methods Efficiently prepared virus-free iPS cells [45, 46] and successfully removed the previously introduced transcription factor genes from the obtained iPS cells [45-47]. In the case of using specific small molecule compounds, iPS cells can be obtained efficiently with less foreign gene introduction, which is a big step towards preparing iPS cells without genetic modification[20, 23, 24, 26, 37, 39, 40, 45～48]. In addition, rat and monkey iPS cell lines have also been established [41～43]. The remaining progress is shown in Table 1. These achievements have greatly improved the practical application of iPS cells in clinical practice. A step forward, iPS cell research and application will be expected to become one of the greatest medical biology achievements in the 21st century. 29]. Virus-free iPS cells were efficiently prepared with the aid of transposon-mediated transgenic methods [45, 46] and successfully removed the previously introduced transcription factor genes from the obtained iPS cells [45-47]. In the case of using specific small molecule compounds, iPS cells can be obtained efficiently with less foreign gene introduction, which is a big step towards preparing iPS cells without genetic modification[20, 23, 24, 26, 37, 39, 40, 45～48]. In addition, rat and monkey iPS cell lines have also been established [41～43]. The remaining progress is shown in Table 1. These achievements have greatly improved the practical application of iPS cells in clinical practice. A step forward, iPS cell research and application will be expected to become one of the greatest medical biology achievements in the 21st century. 29]. Virus-free iPS cells were efficiently prepared with the aid of transposon-mediated transgenic methods [45, 46] and successfully removed the previously introduced transcription factor genes from the obtained iPS cells [45-47]. In the case of using specific small molecule compounds, iPS cells can be obtained efficiently with less foreign gene introduction, which is a big step towards preparing iPS cells without genetic modification[20, 23, 24, 26, 37, 39, 40, 45～48]. In addition, rat and monkey iPS cell lines have also been established [41～43]. The remaining progress is shown in Table 1. These achievements have greatly improved the practical application of iPS cells in clinical practice. A step forward, iPS cell research and application will be expected to become one of the greatest medical biology achievements in the 21st century.
Table 1 The history of induced pluripotent stem cells (iPS cells) Table 1 The history of induced pluripotent stem cells (iPS cells)
2 Several key steps and links for preparing iPS cells
At present, the iPS cell preparation process and its identification are shown in Figure 1. In short: a. Isolation and cultivation of host cells; b. Introducing foreign genes into host cells by means of virus (retrovirus, lentivirus or adenovirus) mediated; c. The virus-infected cells are planted on feeder cells and cultured in a special culture system for ES cells. At the same time, corresponding small molecular substances (such as Wnt3a, 5-AZA, BIX-01294, VPA,
TSA, BayK8644, PD0325901 or CHIR99021, etc.) to promote reprogramming; d. A few days later, ES clone-like clones appeared; e. These clones were identified in terms of cell morphology, gene expression profile, epigenetics, teratoma formation and in vitro differentiation. In view of the fact that Liu Shuang et al. [9] have described the general process of preparing iPS cells and their identification in more detail, it is not repeated here. In addition, Takahashi et al. [49] and Park et al. [50] described the detailed steps of preparing mouse and human iPS cells, respectively. This section only reviews some key steps and links in conjunction with the latest research progress of iPS cells.
2.1 Principles for the selection of somatic cell types and the combination of factors required for reprogramming of somatic cells into iPS cells Table 2, Table 3, and Table 4 show that the combination of different transcription factors or the addition of small molecules has been used to transform mice or humans. Of somatic cells are reprogrammed into iPS cells. Yamanaka's group used Oct4, Sox2, c-Myc, and Klf4 transcription factor combinations to induce mouse and human fibroblasts into iPS cells in vitro[11, 12]. After that, different research groups used similar methods. And the combination of different factors confirmed the feasibility of this method. At the same time, it was also found that the reversed cells are not limited to specific cell types and specific differentiation stages. They can be cells derived from any germ layer of the inner, middle and outer germ layers. It can be derived from embryonic cells or from newborns, adults, or even terminally differentiated mature cells (Table 2, Table 3, and Table 4), but cells from different germ layers or cells at different developmental stages are reprogrammed into iPS cells The difficulty is different, the efficiency is different, the required factor combination is different, or the time required to form a clone is different. Jaenisch's group used the newly established drug-inducible system to study the reprogramming of somatic cells into iPS cells and came to the following conclusions: a. Different induction levels of reprogramming factors can induce somatic cells into iPS cells; b. The duration of transcription factor transgene activity is directly related to the efficiency of reprogramming; c. Somatic cells from many different tissues can be reprogrammed; d. Different types of somatic cells require different transcription factor induction levels for reprogramming into iPS cells [45]. In short, any kind of somatic cells (mouse and human) can theoretically be reprogrammed into iPS cells by these transcription factors. Because human blood cells (such as T cells and B cells), fat cells and skin fibroblasts are easy to obtain and have a wide range of sources, such human somatic cells are ideal donor cells and are suitable for establishing "individual specific", "Patient-specific" or "disease-specific" iPS cells.
The combination of transcription factors required for reprogramming of somatic cells into iPS cells needs to be flexibly selected according to the type of somatic cells and their corresponding transcription factor expression levels or additional small molecule compounds added (Table 2, Table 3, and Table 4). Generally, the combination of Oct4, Sox2, c-Myc, Klf4 or the combination of Oct4, Sox2, Klf4 can reprogram somatic cells into iPS cells, but the reprogramming efficiency of the latter combination is much lower than that of the former (Table 2, Table 3 and Table 4). When using small molecule compounds, it can greatly improve the reprogramming efficiency and/or reduce the number of transcription factors used [20, 23, 24, 26, 37, 39, 40], and increase the number of transcription factors used (Oct4, Sox2, Nanog, Lin28, c-Myc and Klf4) can also greatly improve the efficiency of reprogramming [61]. Another example is the two genes Oct4 and Klf4. Even only one gene of Oct4 can transform neural stem cells or precursor cells into iPS cells. This is mainly due to the high expression of Sox2, c-Myc and Klf4 in these cells [20, 22, 39, 59], and when the small molecule compounds BIX or PD0325901 and CHIR99021 are added to the culture medium, the reprogramming efficiency can be significantly improved [20, 39]. When the two factors of Oct4 and Klf4 are combined with the small molecule compounds BIX and Bay, mouse fibroblasts can be induced into iPS cells with high efficiency. At this time, BIX and Bay can make up for the loss of exogenous Sox2. Reprogramming of fibroblasts into iPS cells requires at least three factors, Oct4, Sox2 and Klf4 [40]. Mouse mature B cells need to be transformed into iPS cells by introducing five genes Oct4, Sox2, Klf4, c-Myc and C/EBPa[19], and when 5-AZA, Oct4, Sox2, Klf4 and c are added to the culture medium -Myc four factors can also induce mature B cells into iPS cells [23]. 2.2 Gene introduction methods Currently, retroviruses [11], lentiviruses [13], adenoviruses [34] and transposons [45, 46] can introduce transcription factor-corresponding genes into somatic cells, and then reprogram them into iPS cells. Recently, both Hochedlinger and Yamanaka groups introduced transcription factor genes into somatic cells through adenovirus-mediated transgenic methods, and then transiently expressed these genes to obtain iPS cells without viral vector integration. The research results of these two groups showed that reprogramming somatic cells into iPS cells only requires the transient expression of transcription factor genes in somatic cells to achieve the goal, without the need for viral vectors to be integrated into the host cell genome. The efficiency of induction into iPS cells is much lower than that of retroviruses and lentiviruses [34,35]. The theoretical basis for reprogramming somatic cells into iPS cells in a non-integrated manner is: the transcription factor is transferred through retrovirus-mediated methods. Tests on iPS cells obtained by gene introduction into somatic cells found that the expression level of transcription factor transgene was very low or the exogenous transgene was completely silenced, while the endogenous transcription factor gene was activated and maintained a high expression level. At this time, the iPS cell is pluripotent. It is maintained by the expression of endogenous transcription factors. So far, exogenous transcription factor transgenes have fulfilled their mission without expression [15,35].
Initially, each retroviral vector or lentiviral vector only carried one transcription factor gene, and the following strategies were adopted to produce virus and infect cells.
There are two options for retrovirus production:
One is the "direct mixing" scheme, that is, a mixture containing 4 or more factor plasmids is used to transfect packaging cells to obtain a virus mixture carrying 4 or more factor genes, and then the virus mixture is used to infect the cells;
The second is to adopt the "divide first and then mix" scheme, that is, first produce viruses carrying each factor gene, and then mix the collected virus supernatant in equal proportions to infect target cells. Lentivirus production can only take
"Separate first and mix later" plan. Recently, an adenoviral vector carrying 3 transcription factor genes (the genes are connected by 2A sequence) [35], an adenoviral vector carrying 4 transcription factor genes [34] and a lentiviral vector [63～65] This will make the process of virus production and cell infection easier. In addition, the four transcription factor genes were cloned into the same vector, and transposon expression vectors were successfully constructed [45,46].
3 The advent of patient- or disease-specific iPS cells In 2008, Eggan’s team extracted skin cells from patients with familial amyotrophic lateral sclerosis (ALS), and used iPS cell technology to reprogram the patient’s somatic cells into " "Disease-specific" human iPS cells are iPS cells derived from patient cells, confirming that the iPS cells are similar to human ES cells in terms of clonal morphology, cell cycle, specific surface antigen expression, stem cell marker gene expression and EBs formation. , And further differentiated into motor neurons by the iPS cells in vitro, and motor neurons are cells damaged in ALS, which indicates that it is possible to "tailor-tail" motor neurons for patients in the future for personalized treatment. The ultimate goal of this approach is to use such "disease-specific" iPS cells to prepare genetically matched healthy cells and use them to replace diseased cells. However, before this method is safely used in humans, there are still Many obstacles need to be overcome [28]. At the same time, patient-specific iPS cells will be an important tool for studying the mechanism of ALS-like diseases and screening drugs to prevent neuronal degeneration. In most cases, ALS is the result of a complex interaction between genetics and environmental factors. This makes it very difficult to use cell culture to study this disease in the laboratory, and the iPS cells from patients with genetic mutations (the mutations make carriers of the mutation more likely to suffer from the disease) happen to be associated with the disease in individual patients. The "numerous" genetic information. Undoubtedly, this result is good news for the study of ALS, and it also means another important step towards the goal of using iPS cells to treat human diseases. In 2008, Daley's team obtained skin fibroblasts or mesenchymal cells derived from osteosarcoma from patients with a series of genetic diseases, and used retrovirus-mediated methods to integrate Oct4, Sox2, Klf4, c-Myc or Oct4, Sox2, Klf4 or Oct4, Sox2, Klf4, c-Myc, Nanog transcription factors were introduced into these cells, and a series of "disease-specific" human iPS cells were successfully obtained [29]. The advent of iPS cell technology lays the foundation for the establishment of “individual-specific”, “patient-specific” or “disease-specific” human iPS cells and the realization of “individualized” drug efficacy and ADME/Tox evaluation. Set a good foundation. At the same time, "patient-specific" or "disease-specific" human iPS cells can also be used to study the pathogenesis of specific diseases (such as human ALS).
4 Prospects for iPS cells without genetic modification The future of iPS cells is to develop safe, efficient, and clinically useful therapeutic stem cells. From a safety perspective, current research is gradually approaching. So far, iPS cells can be obtained by introducing transcription factor genes into somatic cells through retrovirus [11], lentivirus [13] and adenovirus-mediated methods [34, 35]. Retrovirus and lentivirus-mediated transgene methods enable viral vectors to integrate into the host genome to achieve stable expression of transgenes, but these two transgene methods are prone to activate oncogenes, and the iPS cells obtained may have toxic side effects. The Hochedlinger group and the Yamanaka group successively introduced transcription factor genes into somatic cells through adenovirus, and transiently expressed these transcription factors to obtain iPS cells without toxic side effects or virus vector integration, because this gene introduction method generally does not cause viral vectors Integration into the host genome is expected to become a safer method for clinical application [34]. Of course, adenoviral vectors may also integrate into the genome, although this possibility is very small. Using virus as a vector to introduce foreign genes will cause genetic modification of cells, which is potentially dangerous. At the same time, this method is troublesome and impractical to operate. The above methods of establishing iPS cells all involve overexpression (or change) of genes, so how do these genetically modified cells restore constant gene expression? In addition, feeder cells, small fragment carrier contamination, and homogeneity of cultured cells in vitro , Epigenetic changes and genome stability all need to be considered. Although the adenovirus-mediated transgenic method realizes that the transient expression of certain factors replaces the permanent integration of genes to obtain virus-free adeno-iPS cells[34, 35], but this method is still not the best method to obtain safe, efficient, and clinically valuable therapeutic iPS cells. The most ideal and practical method is to use small molecule compounds instead of exogenous gene introduction to achieve somatic cell reprogramming to obtain iPS cells. This idea is expected to become a reality in the near future. As shown in Figure 1 and Table 4, the combination of different transcription factor combinations and small molecule compounds can greatly improve the efficiency of mouse and human cells reprogramming into iPS cells. These small molecule compounds play an important role in promoting cell reprogramming. Neural stem cells or precursor cells express Sox2 highly, so Oct4 and Klf4 two factors can induce such cells into iPS cells (Table 2 and Table 4). Generally, three factors Oct4, Sox2, and Klf4 are needed. Reprogram mouse fibroblasts into iPS cells. When the small molecule BIX and Bay are used in combination, the combination of Oct4 and Klf4 can also efficiently induce mouse fibroblasts into iPS cells [40], at this time BIX The combination with Bay can make up for the lack of exogenous Sox2. It can be seen that the combination of transcription factors and small molecule combinations can reduce
The number of recording factors used. In the absence of exogenous c-Myc, the combination of Oct4, Sox2, Klf4 and Wnt3a can significantly increase the efficiency of reprogramming mouse fibroblasts into iPS cells, which indicates that the Wnt signaling pathway promotes reprogramming [26]. As shown in Table 4, using inhibitors PD0325901 and CHIR99021 to inhibit the MAPK (mitogen-activated protein kinase) signaling pathway and GSK3 (glycogen synthase kinase-3) signaling pathway, respectively, can improve the efficiency of reprogramming mouse neural stem cells into iPS cells[39] . It is not difficult to find that activating some signaling pathways (such as Wnt pathway, etc.) and inhibiting some pathways (such as MAPK and GSK3 pathways, etc.) contribute to somatic cell reprogramming. According to the complex signal network determined by cell fate, "cell-based phenotypic assays" and/or "pathway screens" are used, supplemented by other high-throughput Detection or screening methods have screened out specific small molecules or natural products. They can "specifically" promote the self-renewal, survival, proliferation or differentiation of "specific" stem cells/precursor cells, or "specifically" dedifferentiate mature cells into Stem cells/precursor cells, etc. These achievements are based on humans' in-depth understanding of the main signal pathways [66]. For example, TWS119 and diaminopyrimidine compounds (diaminopyrimidine compounds) can respectively induce mouse ES cells to differentiate into nerve cells[67] or cardiomyocytes[68], 2,6,9- ternary substituted purine (purmorphamine, hedgehog signaling pathway) Agonist) can induce mesenchymal stem cells into osteoblasts[69,70], and 2, 6- Binary replacement purine (reversine) can dedifferentiate skeletal muscle cells into some precursor cells, and further induce differentiation into osteoblasts or adipocytes, etc.[71]. These functional non-peptide/peptide small molecules and natural products have the characteristics of small molecular weight, simple structure, low production cost, easy absorption and stable physiological activity. In the near future, some small molecules and natural products are expected As a medicine for tissue repair and regeneration. The above information tells us that somatic cells can be reprogrammed into iPS cells using only small molecule compounds. However, at present, the complex molecular mechanism of somatic cell reprogramming into iPS cells is still poorly understood. To achieve the above goals, it is necessary to improve the ability to artificially manipulate somatic cell reprogramming and to better understand the reprogramming of somatic cells into iPS cells. Regulation mechanism. Any life activity is determined by a complex signal regulation network formed by the interaction of genetic regulation and epigenetic regulation. Somatic cell reprogramming into iPS cells is no exception. As long as we have a comprehensive understanding of this regulatory network, we can discover some Small molecule compounds to achieve the above purpose. The Jaenisch group and Hochedlinger group established a drug-inducible system to better study the mechanism of somatic cell reprogramming into iPS cells. This system is a new, predictable and reproducible research platform[30～33] . With the help of this system, it is helpful to expedite the elucidation of the molecular mechanism of somatic cell reprogramming and discover more small molecule compounds, which greatly shortens the development cycle of iPS cells from theoretical research to clinical application. A drug-inducible system has been established, which is a new, predictable and reproducible research platform [30-33]. With the help of this system, it is helpful to expedite the elucidation of the molecular mechanism of somatic cell reprogramming and discover more small molecule compounds, which greatly shortens the development cycle of iPS cells from theoretical research to clinical application. A drug-inducible system has been established, which is a new, predictable and reproducible research platform [30-33]. With the help of this system, it is helpful to expedite the elucidation of the molecular mechanism of somatic cell reprogramming and discover more small molecule compounds, which greatly shortens the development cycle of iPS cells from theoretical research to clinical application.
5 Induced differentiation of iPS cells in vivo and in vitro Like human and mouse ES cells, iPS cells implanted under the skin of immunodeficient mice can form teratomas composed of disorderly arrangement of cells derived from the endoderm, neutral and ectoderm at the injection site[11-13, 29]. When mouse iPS cells are injected into mouse blastocysts, they can develop together with recipient cells to form various tissues including the germline to form chimeras [11, 22, 24, 34, 39, 52, 53], At the same time, individuals developed entirely from mouse iPS cells can also be obtained through chimera technology or tetraploid complementation [19,22,24,25,34,39,52,54]. Similarly, iPS cells can differentiate into a multicellular structure containing three germ layer-derived cells in vitro under certain culture conditions, namely EBs, in which ectoderm cells such as β-Ⅲ tubulin-positive neurons and Tuj1-positive nerves can be detected. Cells, Nestin positive cells and astrocytes, mesodermal cells such as CD34 positive cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells and adipocytes, endoderm cells such as AFP (alpha-fetoprotein) positive cells and TROMA-I positive The trophoblastic ectoderm cells etc. [11,12,15,22,32,37,38,62,72]. At the same time, researchers have successfully induced iPS cells to differentiate into neural precursor cells in vitro[18], Functional mature nerve cells, such as motor neurons[28] and dopaminergic neurons[12, 18, 37, 38], astrocytes[18], hematopoietic precursor cells[14], hematopoietic cells[73 ], pancreatic cells and hepatocytes [20], insulin-secreting β cells [74], cardiomyocytes [12, 37, 38, 73, 75～77], smooth muscle cells [73, 76, 78], vascular endothelial cells [ 73,76], pancreatic cells [37] and cochlear hair cells [79], etc. The cells (including precursor cells) induced and differentiated by iPS cells in vitro have shown certain efficacy in the treatment of corresponding diseases. Intrauterine transplantation of neural precursor cells induced and differentiated by iPS cells in vitro 13. After 5 days of fetal rat brain, it can further differentiate into glial cells and various types of neurons (including glutaminergic neurons, GABAergic neurons and catecholaminergic neurons) in the body, and these nerve cells are functionally integrated Enter the host brain and show the activity of mature neurons [18]. At the same time, transplanting dopaminergic neurons induced and differentiated from mouse iPS cells into the brain of a rat model of Parkinson's disease can effectively alleviate the symptoms of the rat's disease and improve its behavior after a period of time [18]. In addition, hematopoietic precursor cells, endothelial precursor cells, mature endothelial cells and cochlear hair cells derived from iPS cells have been successfully used to treat sickle cell anemia [14], hemophilia A [44] and treat neurosensory hearing disorders, respectively [79]. In short, the above in vivo and in vitro experiments fully proved the multi-directional differentiation potential of mouse and human iPS cells. iPS cell technology provides a realistic possibility for each individual to tailor iPS cells[28, 29, 80～83], iPS cells and their derivatives as seed cells for cell replacement therapy have shown good application prospects[14, 18, 79], and the data obtained from the in vitro directed differentiation experiment of iPS cells determines its further clinical application. However, it must be noted that since the current regulatory mechanisms for the self-renewal and differentiation of stem cells (including iPS cells, ES cells and adult stem cells) are still poorly understood, so far humans have not been able to convert stem cells (including iPS cells, iPS cells, ES cells and adult stem cells) are induced to differentiate into any functional cells of interest in vitro. Many ES cell differentiation models in vitro have been established to obtain different types of terminally differentiated cells, but these models have many problems: for example, ES cells cannot differentiate in a single direction or ES cells cannot be differentiated according to our intentions, so they are obtained after induced differentiation. They are often mixed cells. How to accurately control the ES cell differentiation behavior and optimize the induced differentiation technology circuit to achieve the fully directed differentiation of ES cells is a scientific proposition for further discussion. The key to breaking through the above theoretical and technical bottlenecks is to use a suitable model system to clarify the regulatory network that determines cell type specialization, tissue formation, and organogenesis during mammalian development. As humans continue to deepen their understanding of the differentiation and regulation mechanisms of stem cells (including iPS cells, ES cells, and adult stem cells), more types of human cells will be derived from human iPS cells, which will be useful for cell replacement therapy, new drug screening and evaluation, and Other uses provide a large number of cell sources-seed cells.
6 Outlook
The results of iPS cell research are undoubtedly a milestone in the field of stem cell and developmental biology research. It has achieved a series of breakthroughs in a short period of time. It is foreseeable that one day iPS cells will be used in clinics to solve various diseases faced by humans. , But before obtaining safe, efficient, practical, and clinically valuable therapeutic iPS cells, there are still many problems, bottlenecks that need to be broken, and areas that require in-depth research: a. Analyze the molecular mechanism that induces reprogramming of somatic cells into iPS cells, b. To study the regulation mechanism of the biological characteristics and behaviors of iPS cells (such as self-replication, proliferation and differentiation, etc.) and the in vitro induced differentiation mechanism of iPS cells, c. Improve the efficiency of iPS cell preparation, d. Fully evaluate the safety of clinical application of iPS cells, e. Establish an efficient, safe, and practical method for preparing human iPS cells, that is, establishing strategies and methods for preparing iPS cells without genetic modification on the basis of elucidating the mechanism of somatic cell reprogramming into iPS cells (such as using only some small molecules to make human cells) Reprogram as iPS cells), f. On the basis of the previous study, explore a simple technical route and method for preparing "individual-specific" or "disease-specific" therapeutic human iPS cells, and so on.
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