Somatic Cell Reprogramming (Part Three)

2.2. The replacement system iPS cell inducing factor and mechanism

In order to avoid the risk of tumorigenesis caused by the activation of c-Myc in i PS cells, members of the Myc family, L-Myc and N-Myc, can effectively replace c-Myc to induce human and mouse i PS cells, of which the ability of i PS cells to form chimeric mice with germline transmission can be improved, and the resulting mice do not develop tumors. The pluripotency-related factor Glis1 can also replace c-Myc to promote the reprogramming of human and mouse iPS cells, and the resulting chimeras produced by the mouse iPS cells also have the ability of germ line transmission. Glis1 promotes the induction of iPS cells by affecting a variety of reprogramming pathways, including the expression of genes Myc, Nanog, Lin28, Wnt, Essrb and the MET process.

In addition to being replaced by Klf1, Klf2 and Klf5, Klf4 can also be replaced by the orphan nuclear receptor Esrrb and the OS, two factors to achieve the induction of iPS cells. Esrrb mediates reprogramming by up-regulating pluripotent cell-specific genes. The bone morphogenetic protein Bmp4 can also replace Klf4 to promote reprogramming, and its mechanism of action is mainly to promote the MET process. Nanog and Lin28 can replace c-Myc and Klf4 to obtain human iPS cells. Nanog is one of the important factors to maintain the pluripotency of mouse embryonic stem cells. It cooperates with transcription factors such as Oct4 and Sox2 to regulate the pluripotency network, RNA binding protein Lin28 can indirectly regulate the expression of c-Myc to complete reprogramming.

Sox2 can be replaced by Sox1, Sox3, Sox15 and Sox18 of the Sox family. Rcor2 can effectively replace the exogenous Sox2 in the induction of mouse and human iPS cells, and the ogenogen factor Obox1 can replace Sox2 to obtain iPS cells. Obox1 can regulate the expression of cell cycle related genes, slow down the excessive proliferation of reprogrammed cells, and promote the MET process to promote somatic cell reprogramming. The ectoderm cell lineage specialization factor GMNN can also replace Sox2 to obtain human iPS cells. The histone variants TH2A and TH2B, which are abundantly expressed in oocytes, can replace Sox2 and c-Myc to promote the reprogramming of iPS cells. The mechanism is to enrich the X chromosome to inhibit X chromosome inactivation.

The orphan nuclear receptors Nr5a1 and Nr5a2 can replace Oct4 to induce iPS cells, respectively. The mechanism is to activate endogenous Oct4 expression. Oct4 can also be replaced by E-cadherin, the main regulator of epithelial cells. Overexpression of E-cadherin can affect the nuclear localization of β-catenin, thereby promoting the expression of pluripotency genes. In the induction of human iPS cells, the pluripotency-related factor TCL-1A can replace Oct4 and OM only to complete the reprogramming of human fibroblasts. The cells are similar and have the ability to differentiate into three germ layers. The transcription factor Brn4 can also replace Oct4 due to its homologous POU domain.

As the mechanism of each inducing factor in the induction process of iPS cells was gradually elaborated, the induction system in which the classic OSKM four factors were completely replaced also gradually appeared. Studies have shown that Sox2 can initiate the expression of pluripotency genes, including Sall4, Esrrb and Lin28, among which Sall4 can activate other pluripotency genes including Oct4. Therefore, two induction systems that completely replace OSKM are reported: Sall4, Esrrb, Lin28 and Dppa2 or Nanog composed of four factors can complete the induction of iPS cells. However, the induction efficiency of these two systems is low. The six-factor induction system that completely replaces OSKM includes Glis1, Sall4, Lrh1, Jdp2, Jhdm1b, and Id1, which can obtain iPS cells more efficiently.

2.3. The chemical induction system of iPS cells and mechanism

Since exogenous reprogramming factors are easily integrated into the cell genome, and multiple reprogramming factors are involved in the regulation of cancer-related signaling pathways, minimizing the number of transcription factors in the induction system is the first step to improve the biological safety of iPS cells. The use of small molecular compounds instead of reprogramming factors is one of the research directions for improving the safety of iPS cells and optimizing iPS cell technology. The establishment of a complete induction system using small molecule chemicals has revealed more signaling pathways and epigenetic mechanisms related to induced pluripotency.

Histone deacetylase inhibitors (HDACi) Valproic acid (VPA) can effectively replace c-Myc to complete the reprogramming of mouse fibroblasts, and can even replace c-Myc and Klf4 Reprogramming of human fibroblasts. Vitamin C can be reprogrammed with histone demethylase Jhdm1a instead of Klf4, c-Myc and Jhdm1b instead of Sox2, Klf4, c-Myc Inhibiting TGF-β signaling can activate the expression of endogenous Nanog to replace Sox2 and c-Myc. Since the activation of TGF-β plays an important role in the ME differentiation process, inhibiting TGF-β can replace Sox2 induction system. Protein methyltransferase inhibitor BIX01294 and calcium channel agonist BayK8644 can also effectively replace Sox2. Although there have been reports of many systems in which small molecule chemicals are used instead of transcription factors for induction, there has been no progress in screening small molecule compounds to replace Oct4. Using a small molecule combination system of TGF-β inhibitors, HDAC inhibitors, MEK inhibitors and phosphoinositide-dependent kinase PDK1a. Human iPS cells can be induced with a single transcription factor Oct4.

Although both SCNT and iPS cells can bring the cells to a pluripotent state, the ways to achieve pluripotency are different. iPSC technology is to reprogram cells into pluripotent cells similar to ES cells using certain transcription factors or small molecular compounds, while SCNT technology is to transfer donor cells to enucleated oocytes to achieve that donor cells are reprogrammed to a totipotent state similar to fertilized eggs.

The reprogramming speed of SCNT and iPSC technology is also very different. The SCNT reprogramming process occurs very quickly and can be completed within a few hours. The rapid histone replacement driven by egg histones may be the reason for this rapid reprogramming. In contrast, the process of iPS cell reprogramming is relatively slow, and the ectopic expression of exogenous inducing factors first causes a gradual change in cell morphology, and then the expression of pluripotency markers such as alkaline phosphatase and SSEA-1 Before the gradual increase, the expression of somatic cell-specific genes decreased. After a few days or even weeks, the stable expression of endogenous Oct4 and Nanog indicates the completion of the reprogramming process.


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